Human neutralizing monoclonal antibodies to human immunodeficiency virus

ABSTRACT

The present invention describes human monoclonal antibodies which immunoreact with and neutralize human immunodeficiency virus (HIV). Also disclosed are immunotherapeutic and diagnostic methods of using the monoclonal antibodies, as well as cell line for producing the monoclonal antibodies.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Contract No. AI33292 by The National Institutes of Health. The government has certainrights in the invention.

REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. Ser. No. 08/178,302,filed Jan. 6, 1994, now abandoned, which is a U.S. national stage filingpursuant to 35 USC 371 based on PCT application Ser. No. US93/09328,filed Sep. 30, 1993, which is a continuation-in-part of U.S. Ser. No.07/954,148, filed Sep. 30, 1992, now abandoned.

TECHNICAL FIELD

The present invention relates generally to the field of immunology andspecifically to human monoclonal antibodies which bind and neutralizehuman immunodeficiency virus (HIV).

BACKGROUND

1. HIV Immunotherapy

HIV is the focus of intense studies as it is the causative agent foracquired immunodeficiency syndrome (AIDS). Immunotherapeutic methods areone of several approaches to prevention, cure or remediation of HIVinfection and HIV-induced diseases. Specifically, the use ofneutralizing antibodies in passive immunotherapies is of centralimportance to the present invention.

Passive immunization of HIV-1 infected humans using human seracontaining polyclonal antibodies immunoreactive with HIV has beenreported. See for example, Jackson et al., Lancet, Sep. 17: 647-652,(1988); Karpas et al., Proc. Natl. Acad. Sci., USA, 87: 7613-7616(1990).

Numerous groups have reported the preparation of human monoclonalantibodies that neutralize isolates in vitro. The described antibodiestypically have immunospecificities for epitopes on the HIV glycoproteingp120 or the related external surface envelope glycoprotein gp120 or thetransmembrane glycoprotein gp41. See, for example Levy, Micro. Rev., 57:183-289 (1993); Karwowska et al., Aids Research and Human Retroviruses,8: 1099-1106 (1992); Takeda et al., J. Clin. Invest., 89: 1952-1957(1992); Tilley et al., Aids Research and Human Retroviruses, 8: 461-467(1992); Laman et al., J. Virol., 66: 1823-1831 (1992); Thali et al., J.Virol., 65: 6188-6193 (1991); Ho et al., Proc. Natl. Acad. Sci. USA, 88:8949-8952 (1991); D'Souza et al., AIDS, 5: 1061-1070 (1991); Tilley etal., Res. Virol., 142: 247-259 (1991); Broliden et al., Immunol., 73:371-376 (1991); Matour et al., J. Immunol., 146: 4325-4332 (1991); andGorny et al., Proc. Natl. Acad. Sci., USA, 88: 3238-3242 (1991).

To date, none of the reported human monoclonal antibodies have beenshown to be effective in passive immunization therapies. Further, asmonoclonal antibodies, they all each react with an individual epitope onthe HIV envelope glycoprotein, gp120 or gp160. The epitope against whichan effective neutralizing antibody immunoreacts has not been identified.

There continues to be a need to develop human monoclonal antibodypreparations with significant HIV neutralization activity. In addition,there is a need for monoclonal antibodies immunoreactive with additionaland diverse neutralizing epitopes on HIV gp120 and gp41 in view ofrecent studies suggesting that gp120 and gp41 are involved in bothbinding of the HIV virus to the cell as well as in post binding eventsincluding envelope shedding and cleavage. See, for review, Levy, Micro.Rev., 57: 183-289 (1993). Additional (new) epitope specificities arerequired because, upon passive immunization, the administered patientcan produce an immune response against the administered antibody,thereby inactivating the particular therapeutic antibody.

2. Human Monoclonal Antibodies Produced From Combinatorial PhagemidLibraries

The use of filamentous phage display vectors, referred to as phagemids,has been repeatedly shown to allow the efficient preparation of largelibraries of monoclonal antibodies having diverse and novelimmunospecificities. The technology uses a filamentous phage coatprotein membrane anchor domain as a means for linking gene-product andgene during the assembly stage of filamentous phage replication, and hasbeen used for the cloning and expression of antibodies fromcombinatorial libraries. Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366 (1991). Combinatorial libraries of antibodies have beenproduced using both the cpVIII membrane anchor (Kang et al., supra) andthe cpiii membrane anchor. Barbas et al., Proc. Natl. Acad. Sci., USA,88: 7978-7982 (1991).

The diversity of a filamentous phage-based combinatorial antibodylibrary can be increased by shuffling of the heavy and light chain genes(Kang et al., Proc. Natl. Acad. Sci., USA, 88: 11120-11123 (1991)), byaltering the CDR3 regions of the cloned heavy chain genes of the library(Barbas et al., Proc. Natl. Acad. Sci., USA, 89: 4457-4461 (1992)), andby introducing random mutations into the library by error-pronepolymerase chain reactions (PCR) [Gram et al., Proc. Natl. Acad. Sci.,USA, 89: 3576-3580 (1992)].

Filamentous phage display vectors have also been utilized to producehuman monoclonal antibodies immunoreactive with hepatitis B virus (HBV)or HIV antigens. See, for example Zebedee et al., Proc. Natl. Acad.Sci., USA, 89: 3175-3179 (1992); and Burton et al., Proc. Natl. Acad.Sci., USA, 88: 10134-10137 (1991), respectively. None of the previouslydescribed human monoclonal antibodies produced by phagemid vectors thatare immunoreactive with HIV have been shown to neutralize HIV.

In particular, none of the previously-described human monoclonalantibodies produced by phagemid vectors are capable of neutralizing amajority of the field isolates of HIV. It is believed that certain ofthe antibodies described herein are particularly effective atneutralizing HIV because the antibodies immunoreact with an importantantigenic determinant present on "mature" gp120 and not present on theHIV precursor protein gp160.

BRIEF DESCRIPTION OF THE INVENTION

Methods have now been discovered using the phagemid vectors to identifyand isolate from combinatorial libraries human monoclonal antibodiesthat neutralize HIV, and allow the rapid preparation of large numbers ofneutralizing antibodies of completely human derivation. The identifiedneutralizing antibodies define new epitopes on the HIV gp120 and gp41glycoproteins, thereby increasing the availability of newimmunotherapeutic human monoclonal antibodies.

The invention provides human monoclonal antibodies that neutralize HIV,and also provides cell lines used to produce these monoclonalantibodies.

Also provided are amino acid sequences which confer neutralizationfunction to the antigen binding domain of a monoclonal antibody, andwhich can be used immunogenically to identify other antibodies thatspecifically bind and neutralize HIV. The monoclonal antibodies of theinvention find particular utility as reagents for the diagnosis andimmunotherapy of HIV-induced disease.

A major advantage of the monoclonal antibodies of the invention derivesfrom the fact that they are encoded by a human polynucleotide sequence.Thus, in vivo use of the monoclonal antibodies of the invention fordiagnosis and immunotherapy of HIV-induced disease greatly reduces theproblems of significant host immune response to the passivelyadministered antibodies which is a problem commonly encountered whenmonoclonal antibodies of xenogeneic or chimeric derivation are utilized.

An additional major advantage of a preferred group of monoclonalantibodies described herein derives from the fact that they immunoreactwith a unique determinant present on mature HIV glycoprotein gp120. Thisclass of antibodies is particularly effective at neutralizing fieldisolates of HIV.

In one embodiment, the invention contemplates a human monoclonalantibody capable of immunoreacting with human immunodeficiency virus(HIV) glycoprotein gp120 and neutralizing HIV. A preferred humanmonoclonal antibody has the binding specificity of a monoclonal antibodycomprising a heavy chain immunoglobulin variable region amino acidresidue sequence selected from the group consisting of SEQ ID Nos 66,67, 68, 70, 72, 73, 74, 75, 78 and 97.

In a particularly preferred embodiment, the invention describes a humanmonoclonal antibody capable of immunoreacting with humanimmunodeficiency virus (HIV) glycoprotein gp120 and neutralizing HIV,wherein the monoclonal antibody has the capacity to reduce HIVinfectivity titer in an in vitro virus infectivity assay by 50% at aconcentration of less than 700 nanograms (ng) of antibody per milliliter(ml).

Preferably, an anti-gp120 monoclonal antibody of this invention bindsmature gp120 preferentially over HIV precursor glycoprotein gp160. Morepreferably, an anti-gp120 monoclonal antibody binds to a V1/V2 loopdeficient-variant gp120 substantially less than native gp120, therebydefining a important epitope for the antibody. Human monoclonalantibodies having these properties are particularly useful atneutralizing field isolates, and therefore provide useful informationregarding the immunocompetence of an immune response in HIV-infectedpatients.

Therefore, the invention provides for a screening method to determinewhether HIV-infected patients contain antibodies of the class thatneutralize field isolates. The method for determining immunocompetenceof a human anti-human immunodeficiency virus (HIV) antibody in a samplecomprises the steps of:

(1) contacting a sample believed to contain a human anti-HIV antibodywith a diagnostically effective amount of the above-described anti-gp120monoclonal antibody in a competition immunoreaction admixture containingmature gp120 in the solid phase;

(2) maintaining the competition immunoreaction admixture underconditions sufficient for the monoclonal antibody to bind with the gp120in the solid phase and form a solid phase immunoreactant; and

(3) detecting the amount of the immunoreactant present in the solidphase, and thereby the immunocompetence of any human anti-HIV antibodyin the sample.

Another preferred human monoclonal antibody has the binding specificityof a monoclonal antibody comprising a light chain immunoglobulinvariable region amino acid residue sequence selected from the groupconsisting of SEQ ID Nos 95, 96, 97, 98, 101, 102, 103, 104, 105, 107,110, 115, 118, 121, 122, 124 and 132.

In a further embodiment, the invention contemplates a human monoclonalantibody capable of immunoreacting with human immunodeficiency virus(HIV) glycoprotein gp41 and neutralizing HIV. A preferred humanmonoclonal antibody has the binding specificity of a monoclonal antibodycomprising a heavy chain immunoglobulin variable region amino acidresidue sequence selected from the group consisting of SEQ ID Nos 142,143, 144, 145 and 146. Another preferred human monoclonal antibody hasthe binding specificity of a monoclonal antibody comprising a lightchain immunoglobulin variable region amino acid residue sequenceselected from the group consisting of SEQ ID NOs 147, 148, 149, 150 and151.

In another embodiment, the invention describes a polynucleotide sequenceencoding a heavy or light chain immunoglobulin variable region aminoacid residue sequence portion of a human monoclonal antibody of thisinvention. Also contemplated are DNA expression vectors containing thepolynucleotide, and host cells containing the vectors andpolynucleotides of the invention.

The invention also contemplates a method of detecting humanimmunodeficiency virus (HIV) comprising contacting a sample suspected ofcontaining HIV with a diagnostically effective amount of the monoclonalantibody of this invention, and determining whether the monoclonalantibody immunoreacts with the sample. The method can be practiced invitro or in vivo, and may include a variety of methods for determiningthe presence of an immunoreaction product.

In another embodiment, the invention describes a method for providingpassive immunotherapy to human immunodeficiency virus (HIV) disease in ahuman, comprising administering to the human an immunotherapeuticallyeffective amount of the monoclonal antibody of this invention. Theadministration can be provided prophylactically, and by a parenteraladministration. Pharmaceutical compositions containing one or more ofthe different human monoclonal antibodies are described for use in thetherapeutic methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure:

FIG. 1 illustrates the sequence of the double-stranded synthetic DNAinserted into Lambda Zap to produce a Lambda Hc2 expression vector. Thepreparation of the double-stranded synthetic DNA insert is described inExample 1a2). The various features required for this vector to expressthe V_(H) -coding DNA homologs include the Shine-Dalgarno ribosomebinding site, a leader sequence to direct the expressed protein to theperiplasm as described by Mouva et al., J. Biol. Chem., 255: 27, 1980,and various restriction enzyme sites used to operatively link the V_(H)homologs to the expression vector. The V_(H) expression vector sequencealso contains a short nucleic acid sequence that codes for amino acidstypically found in variable regions heavy chain (V_(H) backbone). ThisV_(H) backbone is just upstream and in the proper reading as the V_(H)DNA homologs that are operatively linked into the Xho I and Spe Icloning sites. The sequences of the top and bottom strands of thedouble-stranded synthetic DNA insert are listed respectively in SEQ IDNO 1 and SEQ ID NO 2. The ten amino acid sequence comprising thedecapeptide tag is listed in SEQ ID NO 5. The synthetic DNA insert isdirectionally ligated into Lambda Zap II digested with the restrictionenzymes Not 1 and Xho I to form Lambda Hc2 expression vector.

FIG. 2 illustrates the major features of the bacterial expression vectorLambda Hc2 (V_(H) expression vector). The orientation of the insert inLambda Zap II is shown. The V_(H) DNA homologs are inserted into the XhoI and Spe I cloning sites. The read through transcription produces thedecapeptide epitope (tag) that is located just 3' of the cloning site.The amino acid residue sequence of the decapeptide tag and the Pel Bleader sequence/spacer are respectively listed in SEQ ID NO 5 and 6.

FIG. 3 illustrates the sequence of the double-stranded synthetic DNAinserted into Lambda Zap to produce a Lambda Lc2 expression vector. Thevarious features required for this vector to express the V_(L) -codingDNA homologs are described in FIG. 1. The V_(L) -coding DNA homologs areoperatively linked into the Lc2 sequence at the Sac I and Xho Irestriction sites. The sequences of the top and bottom strands of thedouble-stranded synthetic DNA insert are listed respectively in SEQ IDNO 3 and SEQ ID NO 4. The synthetic DNA insert is directionally ligatedinto Lambda Zap II digested with the restriction enzymes Sac I and Not Ito form Lambda Lc2 expression vector.

FIG. 4 illustrates the major features of the bacterial expression vectorLc2 (V_(L) expression vector). The synthetic DNA sequence from FIG. 3 isshown at the top along with the LacZ promoter from Lambda Zap II. Theorientation of the insert in Lambda Zap II is shown. The V_(L) DNAhomologs are inserted into the Sac I and Xho I cloning sites. The aminoacid residue sequence of the Pel B leader sequence/spacer is listed inSEQ ID NO 7.

FIG. 5 illustrates the dicistronic expression vector, pComb, in the formof a phagemid expression vector.

FIG. 6 illustrates the neutralization of HIV-1 by recombinant Fabs. Thesame supernate preparations were used in p24 and syncytia assays. Thefigures indicate neutralization titers. Refer to Example 3 for detailsof the assay procedures and discussion of the results. The ELISA titersand Fab concentrations were determined as described in Example 2b.

FIG. 7 illustrates the relative affinities of Fab fragments for gp120(IIIB) as illustrated by inhibition ELISA performed as described inExample 2b6). Fabs 27, 6, 29, 2 and 3 are all prototype members of thedifferent groups discussed in Example 9a. Loop 2 is an Fab fragmentselected from the same library as the other Fabs but which recognizesthe V3 loop. The data is plotted as the percentage of maximum binding onthe Y-axis against increasing concentrations (10⁻¹¹ M to 10⁻⁷ M) ofsoluble gp120 on the X-axis.

FIG. 8 illustrates the soluble CD4 competition with Fab fragments forgp120 (IIIB). P4D10 and loop2 are controls. P4D10 is a mouse monoclonalantibody reacting with the V3 loop of gp120 (IIIB). The data, discussedin Example 2b6), is plotted as described in FIG. 7.

FIG. 9 illustrates the neutralization of HIV by purified Fabs preparedas described in Example 3. The results shown are derived from thesyncytia assay using the MN strain. The data is plotted as percent ofinhibition of binding on the Y-axis against increasing Fabconcentrations [0.1 to greater than 10 micrograms/milliliter (μg/ml)] onthe X-axis.

FIGS. 10A-10B illustrate the amino acid residue sequences of variableheavy (V_(H)) domains of Fabs binding to gp120. Seven distinct groupshave been identified as described in Example 9a based on sequencehomology. Identity with the first sequence in a group is indicated bydots. The Fab clone names are indicated in the left hand column. Thecorresponding SEQ ID Nos are indicated in the right hand column. Thesequenced regions from right to left are framework region 1 (FR1),complementary determining region 1 (CDR1), framework region 2 (FR2),complementary determining region 2 (CDR2), framework region 3 (FR3),complementary determining region 3 (CDR3), and framework region 4 (FR4).The five amino-terminal residue sequence beginning with LEQ arises fromthe VH1a while the 5 amino-terminal residue sequence beginning with LEEarises from the VH3a primers. The b11 and b29 sequences are very similarto the b3 group and could be argued to be intraclonal variants withinthat group; they are placed in their own group because of differences atthe V-D and D-J interface.

FIGS. 11A-11B illustrate the amino acid residue sequences of variablelight (V_(L)) domains of Fabs binding to gp120. Refer to FIG. 10 for thedescription of the figure and to Example 9b for analysis of thesequences.

FIGS. 12A-12B illustrate the amino acid residue sequences of V_(L)domains from Fabs binding to gp120 and generated by shuffling the heavychain from clone b12 against a library of light chains (H12-LCn Fabs) asdescribed in Example 10. Note that the new V_(L) sequences havedesignated clone numbers that do not relate to those numbers from theoriginal library. The unique sequences are listed in the SequenceListing from SEQ ID NO 114 to 122. The new V_(L) domain sequences arecompared to that of the original clone b12 V_(L) sequence.

FIGS. 13A-13B illustrate the amino acid residue sequences of V_(H)domains from Fabs binding to gp120 and generated by shuffling the lightchain from clone b12 against a library of heavy chains (L12-HCn Fabs) asdescribed in Example 10. Note that the new V_(H) sequences havedesignated clone numbers that do not relate to those numbers from theoriginal library. The unique sequences are listed in the SequenceListing from SEQ ID NO 123 to 132. The new V_(H) domain sequences arecompared to that of the original clone b12 V_(H) sequence.

FIGS. 14A-14B illustrate, in two figures, FIGS. 14A and 14B, plasmidmaps of the heavy (pTAC01H) and light chain (pTC01) replicon-compatiblechain-shuffling vectors, respectively. Both plasmids are very similar inthe section containing the promoter and the cloning site. Abbreviations:tacPO, tac promoter/operon; 5 histidine amino acid residue tag(histidine) 5-tail; f1IG, intergenic region of f1-phage; stu, stufferfragment ready for in-frame replacement by light and heavy chain,respectively; cat, chloramphenicol transferase gene; bla, b-lactamasegene; ori, origin of replication. The map is drawn approximately toscale.

FIGS. 15A-15B illustrate the nucleotide sequences of the binaryshuffling vectors in two Figures, 15A and 15B. The construction and useof the vectors is described in Example 11. In FIG. 15A, thedouble-stranded nucleotide sequence of the multiple cloning site inlight chain vector, pTC01, is shown. The sequences of the top and bottomnucleotide base strands are listed respectively in SEQ ID NO 8 and SEQID NO 9. The amino acid residue sequence comprising the pelB leaderending in the Sac I restriction site is listed in SEQ ID NO 10. In FIG.15B, the nucleotide sequence of the multiple cloning site in heavy chainvector, pTAC01H, is shown. The sequences of the top and bottomnucleotide base strands are listed respectively in SEQ ID NO 11 and SEQID NO 12. The amino acid residue sequence comprising the pelB leaderending in the Xho I restriction site is listed as SEQ ID NO 13. Theamino acid residue sequence comprising the histidine tail is listed inSEQ ID NO 14. Relevant restriction sites are underlined. tac promoterand ribosome binding site (rbs) are indicated by boxes.

FIG. 16 illustrates the complete set of directed crosses between heavyand light chains of all Fab fragments isolated from the original libraryby panning with gp160 (IIIB) (b1-b27), gp120 (IIIB) (B8-B35), gp120(SF2) (s4-s8), and the loop peptide (p35) assayed by ELISA against IIIB910120 as described in Example 11. Heavy chains are listed horizontallyand light chains are listed vertically. Clones are sorted according tothe grouping established in Example 9. Different groups are separated byhorizontal and vertical lines. A "-" at the intersection of a particularheavy chain and light chain signifies a clear negative (a signal of 3times background or less) for that particular cross, a "+" shows a clearpositive comparable to the original heavy and light chain combination,and a "w" denotes an intermediate value in the ELISA. "": theHCp35/LCp35 combination is negative when gp120 (IIIB) is used, butpositive when assayed with gp120 (IIIB). Identical chains carry the sameidentifier (either *, ¶, §, or ¥).

FIG. 17 illustrates the affinity of antibody-antigen interaction for b12heavy chain crosses with light chains from all pannings analyzed bycompetitive ELISA using soluble IIIB 910120 as competing antigen asdescribed in Example 10. The data is plotted as the percentage ofmaximum binding on the Y-axis against increasing concentrations ofsoluble gp120 (IIIB) (10⁻¹² M to 10⁻⁷ M) on the X-axis.

FIGS. 18A-18B illustrate the amino acid residue sequences of variableheavy (V_(H)) domains of Fabs binding to gp41. The Fab clone names areindicated in the left hand column. The heavy chain sequences of the fiveFabs individually designated DL 41 19, DO 41 11, GL 41 1, MT 41 12 andSS 41 8 have been assigned the respective SEQ ID Nos 142, 143, 144, 145and 146. The sequenced regions from right to left are framework region 1(FR1), complementary determining region 1 (CDR1), framework region 2(FR2), complementary determining region 2 (CDR2), framework region 3(FR3), complementary determining region 3 (CDR3), and framework region 4(FR4).

FIGS. 19A-19B illustrate the amino acid residue sequences of variablelight (V_(L)) domains of Fabs binding to gp41. Refer to FIG. 18 for thedescription of the figure. The light chain sequences of the five Fabsindividually designated DL 41 19, DO 41 11, GL 41 1, MT 41 12 and SS 418 have been assigned the respective SEQ ID NOs 147, 148, 149, 150 and151.

FIG. 20 illustrates the relative binding affinities of b3, b6, and b12for the total envelope glycoproteins (gp160) and for the gp120glycoprotein (gp120) expressed on the surface of COS-1 cells asdetermined by immunoprecipitation and described in Example 6. The signalon the autoradiogram represents the relative amount of envelopeglycoproteins bound with increasing concentrations of Fab (0-150 μg/ml).

FIG. 21 illustrates the neutralization of HIV-1 by b12 IgG1 as assessedusing PHA-stimulated PBMCs as indicator cells and determination ofextracellular p24 as the reporter assay. Refer to Example 5d for detailsof the assay procedures and discussion of the results. The designation,location, and disease status of the virus donors were as follows: ▪, VS(New York, acute), ▾, N70-2 (New Orleans, asymptomatic), ▴, AC (SanDiego, AIDS), , LS (Los Angeles, AIDS), □, NYC-A (New York, unknown),∇, WM (Los Angeles, AIDS), Δ, RA (New York, acute), ⋄, JP (New York,acute). The molecularly cloned HIV-1 virus JR-CSF (♦) and HIV-1 isolateJR-FL (∘) were also assayed for neutralization. The data is plotted as %neutralization on the Y-axis against increasing concentrations of b12IgG1 (0-25 μg/ml) on the X-axis.

FIG. 22 illustrates the reactivity of b12 IgG1 with a panel ofinternational isolates of HIV-1 as described in Example 8. Reactivitywas determined with gp120 isolated from the HIV-1 samples in ELISA withthe b12 IgG1 as described in Example 8. Data is plotted as % b12 IgG1reactivity on the X-axis against clades A-F on the Y-axis. Country namesindicate where the HIV-1 virus was originally isolated. The numbers inparenthesis refer to the number of viruses of each clade examined.Reactivity is designated as so strong () or moderate ().

FIG. 23 illustrates the neutralization of the HXBc2 molecular clone ofHIV-1 LAI by purified Fabs and a monoclonal antibody 110.4 (Mab 110.4)in an envelope complementation assay as described in Example 3c.Neutralization of HXBc2 infectivity is expressed as a decrease inresidual CAT activity. The data is plotted as % residual CAT activity onthe Y-axis and increasing concentrations of Fab and MAb (0.1-20 μg/ml)on the X-axis.

FIG. 24 illustrates the pSG-5 mammalian expression vector as describedin Examples 4a and 4b. Transcription of the heavy or light chain genewhen inserted in the EcoRI site is under the control of the SV40 earlypromoter. Transcriptional termination is signaled by the SV40polyadenylation signal sequence downstream of the heavy chain sequence.The M13 intergenic region allows for the production of single-strandedDNA for nucleotide sequence determination. The amp^(R) gene is forselection of the vector in bacterial cells.

FIGS. 25A and 25B illustrate the nucleotide and amino acid residuesequences of the b12 light chain gene in the pSG-5 mammalian expressionvector described in Example 4b. The b12 light chain has been modifiedfor expression in mammalian cells as described in Example 4b.

FIG. 26 illustrates pEe6HC BM12, the pEE6 mammalian expression vectorwith the b12 IgG1 heavy chain gene that has been modified for antibodyexpression in mammalian cells as described in Example 4d. The VH wasoriginally derived from the Fab b12 and has the same binding specificityas the Fab b12. The pEE6 vector has a human CMV promoter for expressionof the heavy chain, a polyadenylation signal for termination oftranscription, and an ampicillin gene for selection in bacteria.

FIGS. 27A through 27E illustrate the nucleotide sequence of the b12heavy chain VH and constant regions in the pEe6HC BM12 mammalianexpression vector as described Example 4d. The amino acid residuesequence of the b12 heavy chain VH is given. The b12 VH has beenmodified for expression in mammalian cells as described in Example 4d.

FIG. 28 illustrates pEe12 Combo BM12, the pEE12 mammalian expressionvector with b12 IgG1 heavy and light chain genes that have been modifiedfor antibody expression in mammalian cells as described in example 4f.The VH and light chain were originally derived from the Fab b12 and havethe same binding specificity as the Fab b12. The pEE12 vector has ahuman CMV promoter for expression of the light chain, a polylinker toprovide cloning sites, and a polyadenylation signal for termination oftranscription. The vector also contains the GS selectable marker genewhose expression is controlled an SV40 early promoter at the 5' end ofthe GS gene, an intron, and a polyadenylation signal at the 3' end ofthe GS gene. A heavy chain cassette comprising the HCMV promoter,enhancer elements, heavy chain gene, and polyadenylation signal wereremoved from the pEE6 vector and inserted into the pEE12 vector togenerate the combinatorial construct containing both the b12 light andheavy chain genes.

FIGS. 29A through 29S illustrates the nucleotide sequence of the pEE12mammalian expression vector and the b12 IgG1 heavy and light chaingenes, pEe12 Combo BM 12, as described in Example 4f. The VH and lightchain genes have been modified for expression in mammalian cells asdescribed in Example 4.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Amino Acid Residue: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the "L" isomeric form.However, residues in the "D" isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxy terminus of a polypeptide. In keeping withstandard polypeptide nomenclature (described in J. Biol. Chem., 243:3552-59 (1969) and adopted at 37 CFR §1.822(b) (2)), abbreviations foramino acid residues are shown in the following Table of Correspondence:

    ______________________________________    TABLE OF CORRESPONDENCE    SYMBOL    1-Letter 3-Letter       AMINO ACID    ______________________________________    Y        Tyr            tyrosine    G        Gly            glycine    F        Phe            phenylalanine    M        Met            methionine    A        Ala            alanine    S        Ser            serine    I        Ile            isoleucine    L        Leu            leucine    T        Thr            threonine    V        Val            valine    P        Pro            proline    K        Lys            lysine    H        His            histidine    Q        Gln            glutamine    E        Glu            glutamic acid    Z        Glx            Glu and/or Gln    W        Trp            tryptophan    R        Arg            arginine    D        Asp            aspartic acid    N        Asn            asparagine    B        Asx            Asn and/or Asp    C        Cys            cysteine    X        Xaa            Unknown or other    ______________________________________

It should be noted that all amino acid residue sequences representedherein by formulae have a left-to-right orientation in the conventionaldirection of amino terminus to carboxy terminus. In addition, the phrase"amino acid residue" is broadly defined to include the amino acidslisted in the Table of Correspondence and modified and unusual aminoacids, such as those listed in 37 CFR 1.822(b) (4), and incorporatedherein by reference. Furthermore, it should be noted that a dash at thebeginning or end of an amino acid residue sequence indicates a peptidebond to a further sequence of one or more amino acid residues or acovalent bond to an amino-terminal group such as NH₂ or acetyl or to acarboxy-terminal group such as COOH.

Recombinant DNA (rDNA) molecule: A DNA molecule produced by operativelylinking two DNA segments. Thus, a recombinant DNA molecule is a hybridDNA molecule comprising at least two nucleotide sequences not normallyfound together in nature. RDNA'S not having a common biological origin,i.e., evolutionarily different, are said to be "heterologous".

Vector: A RDNA molecule capable of autonomous replication in a cell andto which a DNA segment, e.g., gene or polynucleotide, can be operativelylinked so as to bring about replication of the attached segment. Vectorscapable of directing the expression of genes encoding for one or morepolypeptides are referred to herein as "expression vectors".Particularly important vectors allow cloning of cDNA (complementary DNA)from mRNAs produced using reverse transcriptase.

Receptor: A receptor is a molecule, such as a protein, glycoprotein andthe like, that can specifically (non-randomly) bind to another molecule.

Antibody: The term antibody in its various grammatical forms is usedherein to refer to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantibody combining site or paratope. Exemplary antibody molecules areintact immunoglobulin molecules, substantially intact immunoglobulinmolecules and portions of an immunoglobulin molecule, including thoseportions known in the art as Fab, Fab', F(ab')₂ and F(v).

Antibody Combining Site: An antibody combining site is that structuralportion of an antibody molecule comprised of a heavy and light chainvariable and hypervariable regions that specifically binds (immunoreactswith) an antigen. The term immunoreact in its various forms meansspecific binding between an antigenic determinant-containing moleculeand a molecule containing an antibody combining site such as a wholeantibody molecule or a portion thereof.

Monoclonal Antibody: A monoclonal antibody in its various grammaticalforms refers to a population of antibody molecules that contain only onespecies of antibody combining site capable of immunoreacting with aparticular epitope. A monoclonal antibody thus typically displays asingle binding affinity for any epitope with which it immunoreacts. Amonoclonal antibody may therefore contain an antibody molecule having aplurality of antibody combining sites, each immunospecific for adifferent epitope, e.g., a bispecific monoclonal antibody. Althoughhistorically a monoclonal antibody was produced by immortalization of aclonally pure immunoglobulin secreting cell line, a monoclonally purepopulation of antibody molecules can also be prepared by the methods ofthe present invention.

Fusion Polypeptide: A polypeptide comprised of at least two polypeptidesand a linking sequence to operatively link the two polypeptides into onecontinuous polypeptide. The two polypeptides linked in a fusionpolypeptide are typically derived from two independent sources, andtherefore a fusion polypeptide comprises two linked polypeptides notnormally found linked in nature.

Upstream: In the direction opposite to the direction of DNAtranscription, and therefore going from 5' to 3' on the non-codingstrand, or 3' to 5' on the mRNA.

Downstream: Further along a DNA sequence in the direction of sequencetranscription or read out, that is traveling in a 3'- to 5'-directionalong the non-coding strand of the DNA or 5'- to 3'-direction along theRNA transcript.

Cistron: Sequence of nucleotides in a DNA molecule coding for an aminoacid residue sequence and including upstream and downstream DNAexpression control elements.

Leader Polypeptide: A short length of amino acid sequence at the aminoend of a polypeptide, which carries or directs the polypeptide throughthe inner membrane and so ensures its eventual secretion into theperiplasmic space and perhaps beyond. The leader sequence peptide iscommonly removed before the polypeptide becomes active.

Reading Frame: Particular sequence of contiguous nucleotide triplets(codons) employed in translation. The reading frame depends on thelocation of the translation initiation codon.

B. Human Monoclonal Antibodies

The present invention relates to human monoclonal antibodies which arespecific for, and neutralize human immunodeficiency virus (HIV). In apreferred embodiment of the invention, human monoclonal antibodies aredisclosed which are capable of binding epitopic polypeptide sequences inglycoprotein gp120 of HIV. A further preferred embodiment are humanmonoclonal antibodies capable of binding epitopic polypeptide sequencesin glycoprotein gp 41 of HIV. Also disclosed is an antibody having aspecified amino acid sequence, which sequence confers the ability tobind a specific epitope and to neutralize HIV when the virus is bound bythese antibodies. A human monoclonal antibody with a claimedspecificity, and like human monoclonal antibodies with like specificity,are useful in the diagnosis and immunotherapy of HIV-induced disease.

The term "HIV-induced disease" means any disease caused, directly orindirectly, by HIV. An example of a HIV-induced disease is acquiredautoimmunodeficiency syndrome (AIDS), and any of the numerous conditionsassociated generally with AIDS which are caused by HIV infection.

Thus, in one aspect, the present invention is directed to humanmonoclonal antibodies which are reactive with a HIV neutralization siteand cell lines which produce such antibodies. The isolation of celllines producing monoclonal antibodies of the invention is described ingreat detail further herein, and can be accomplished using the phagemidvector library methods described herein, and using routine screeningtechniques which permit determination of the elementary immunoreactionand neutralization patterns of the monoclonal antibody of interest.Thus, if a human monoclonal antibody being tested binds and neutralizesHIV in a manner similar to a human monoclonal antibody produced by thecell lines of the invention then the tested antibody is consideredequivalent to an antibody of the invention.

It is also possible to determine, without undue experimentation, if ahuman monoclonal antibody has the same (i.e., equivalent) specificity asa human monoclonal antibody of this invention by ascertaining whetherthe former prevents the latter from binding to HIV. If the humanmonoclonal antibody being tested competes with the human monoclonalantibody of the invention, as shown by a decrease in binding by thehuman monoclonal antibody of the invention in standard competitionassays for binding to a solid phase antigen, for example to gp120, thenit is likely that the two monoclonal antibodies bind to the same, or aclosely related, epitope.

Still another way to determine whether a human monoclonal antibody hasthe specificity of a human monoclonal antibody of the invention is topre-incubate the human monoclonal antibody of the invention with HIVwith which it is normally reactive, and then add the human monoclonalantibody being tested to determine if the human monoclonal antibodybeing tested is inhibited in its ability to bind HIV. If the humanmonoclonal antibody being tested is inhibited then, in all likelihood,it has the same, or functionally equivalent, epitopic specificity as themonoclonal antibody of the invention. Screening of human monoclonalantibodies of the invention, can be also carried out utilizing HIVneutralization assays and determining whether the monoclonal antibodyneutralizes HIV.

The ability to neutralize HIV at one or more stages of virus infectionis a desirable quality of a human monoclonal antibody of the presentinvention. Virus neutralization can be measured by a variety of in vitroand in vivo methodologies. Exemplary methods described herein fordetermining the capacity for neutralization are the in vitro assays thatmeasure inhibition of HIV-induced syncytia formation, plaque assays andassays that measure the inhibition of output of core p24 antigen from acell infected with HIV.

As shown herein, the immunospecificity of a human monoclonal antibody ofthis invention can be directed to epitopes that are shared acrossserotypes and/or strains of HIV, or can be specific for a single strainof HIV, depending upon the epitope. Thus, a preferred human monoclonalantibody can immunoreact with HIV-1, HIV-2, or both, and can immunoreactwith one or more of the HIV-1 strains IIIB, MN, RF, SF-2, Z2, Z6, CDC4,ELI and the like strains. In addition, a preferred human monoclonalantibody can immunoreact and neutralize a majority of field isolates ofHIV, as described further herein.

The immunospecificity of an antibody, its HIV-neutralizing capacity, andthe attendant affinity the antibody exhibits for the epitope, aredefined by the epitope with which the antibody immunoreacts. The epitopespecificity is defined at least in part by the amino acid residuesequence of the variable region of the heavy chain of the immunoglobulinthe antibody, and in part by the light chain variable region amino acidresidue sequence. Preferred human monoclonal antibodies immunoreact withthe CD4 binding site of glycoprotein gp120.

Also disclosed is an antibody having a specified amino acid sequence,which sequence confers the ability to bind a specific uniqueneutralizing epitope and to neutralize HIV when the virus is bound bythese antibodies.

A preferred human monoclonal antibody of this invention has the bindingspecificity of a monoclonal antibody comprising a heavy chainimmunoglobulin variable region amino acid residue sequence selected fromthe group of sequences consisting of SEQ ID NOs 66, 67, 68, 70, 72, 73,74, 75, 78 and 97, and conservative substitutions thereof.

Another preferred human monoclonal antibody of this invention has thebinding specificity of a monoclonal antibody having a light chainimmunoglobulin variable region amino acid residue sequence selected fromthe group of sequences consisting of SEQ ID NOs 95, 96, 97, 98, 101,102, 103, 104, 105, 107, 110, 115, 118, 121, 122, 124 and 132, andconservative substitutions thereof.

In a preferred embodiment, a monoclonal antibodies of this inventionexhibits a potent capacity to neutralize HIV. The capacity to neutralizeHIV is expressed as a concentration of antibody molecules required toreduce the infectivity titer of a suspension of HIV when assayed in antypical in vitro infectivity assay, such as is described herein. Amonoclonal antibody of this invention has the capacity to reduce HIVinfectivity titer in an in vitro virus infectivity assay by 50% at aconcentration of less than 700 nanograms (ng) of antibody per milliliter(ml) of culture medium in the assay, and preferably reduces infectivitytiters 50% at a concentration of less than 300 ng/ml, and morepreferably at concentrations less than about 10 ng/ml.

Exemplary and preferred monoclonal antibodies described herein areeffective at 3-700 ng/ml, and therefore are particularly well suited forinhibiting HIV in vitro and in vivo.

Particularly preferred human monoclonal antibodies of this inventionimmunoreact with gp120 in its "mature" form, which form is to bedistinguished from antigenic determinants present on the envelopeprecursor glycoprotein designated gp160. gp160 is processed during virusbiogenesis by cleavage into two polypeptides, gp41 and gp120. "Mature"gp120 refers to the processed protein that is found in mature HIV virusparticles, and can be detected on the surface of HIV-infected cells.

Thus, a preferred antibody of this invention binds mature gp120preferentially over HIV precursor glycoprotein gp160. By "bindspreferentially" is meant that the antibody immunoreacts with (binds)substantially more mature gp120 than gp160 in an immunoreactionadmixture. Substantially more typically indicates that at least greaterthan 50% of the total mass of immunoprecipitated material is gp120, andpreferably indicates that at least greater than 75%, more preferably90%, of the immunoprecipitated material is gp120.

Methods for determining immunoreaction of a subject antibody with gp120or gp160 are well known in the art, and the invention need not be solimited. However, preferred methods for determining the relative amountsof envelope glycoprotein antigens are described in the Examples, andinclude radio-immunoprecipitation (RIP) of cell-surface labeledHIV-infected cells, followed by molecular weight analysis of the labeledproducts by polyacrylamide gel electrophoresis (PAGE).

A preferred human monoclonal antibody also has the ability toimmunoreact with native gp120 and comparatively bind substantially lessof a variant gp120 produced by recombinant DNA methods in which the V1and V2 loops have been deleted. The variant gp120, also referred to aV1/V2 loop deficient-variant gp120, is described in the Examples, and isseen to bind substantially less of a preferred antibody, b12, incomparison to native gp120. The term "native gp120" refers to a maturegp120 protein having a normal amino acid residue sequence instead of avariant protein having selected amino acid residue substitutions ordeletions, such as the V1/V2 loop deficient-variant in which the V1 andV2 loops were deleted. This preferential binding with native gp120compared to the V1/V2 loop deficient-variant identifies an importantepitope defined by a preferred antibody of this invention. Antibodieshaving this binding epitope are particularly effective at neutralizing amajority of field isolates of HIV, as described herein.

The ability to bind "substantially less" V1/V2 loop deficient-variantgp120 than native gp120 can be readily measured using variousimmunoreaction detection methods, although the assay methods describedin Example 5c are particularly preferred. In preferred embodiments,substantially less binding to V1/V2 loop deficient-variant gp120compared to native gp120 is indicated when the comparison is conductedas described as in Example 5c, and the native gp120 exhibits a ratiovalue deviating from the mean of greater than 2.0 and the variantexhibits a ratio value deviating from the mean of less than 0.5.

A particularly preferred human monoclonal antibody of this inventionalso has the capacity to neutralize a majority of field isolates asdisclosed herein. As is well understood, the field (i.e., clinicallyisolated) strains of HIV are typically different to some degreeantigenically from laboratory strains. Therefor, it is well understoodthat useful neutralizing antibodies must immunoreact with, and beneutralizing against, field isolates of HIV. Preferably, the usefulantibody neutralized a large percentage of field isolates, therebyincreasing its effectiveness when new strains are encountered.

The Examples demonstrate that the human monoclonal antibody b12 has theability to neutralize a majority of the field isolates tested. Bymajority is meant that in a representative and diverse collection offield isolates, the antibody is capable of neutralizing at least 50% ofthe strains, and preferably at least 75% of the strains tested. In thiscontext, "neutralizing" means an effect of reducing the HIV infectivitytitre in an in vitro virus infectivity assay as described herein at theantibody concentrations described.

Thus, the invention also contemplates a human monoclonal antibodycapable of immunoreacting with and neutralizing a first preselectedhuman immunodeficiency virus (HIV), such as the laboratory isolate MN orIIIB, that is further capable of immunoreacting with and neutralizingone or more other (i.e., second) strains of HIV, particularly fieldstrains. In this embodiment, supported by the teachings of the Examples,the antibody has the capacity to reduce HIV infectivity titer in an invitro virus infectivity assay of the first HIV strain by 50% at aconcentration of at least less than 700 nanograms (ng) of antibody permilliliter (ml), and has the capacity to reduce HIV infectivity titer ofa second field strain of HIV in the same in vitro virus infectivityassay by 50% at a concentration of less than about 700 nanograms (ng) ofantibody per milliliter (ml). In more preferred embodiments anddepending upon the particular HIV strain, the capacity to reduce secondfield strain infectivity titers by 50% can be exhibited at lowerantibody concentrations, such as below 300 ng/ml.

A particularly preferred antibody is an antibody having the bindingspecificity of the b12 monoclonal antibody described herein. The aminoacid residue sequence of the heavy chain variable region of b12 is shownin SEQ ID NO 66, and the light chain variable region sequence of b12 isshown in SEQ ID NO 97. Still more preferred are human antibodies havingthe binding specificity of the immunoglobulin heavy and light chainpolypeptides produced by ATCC 69079.

Further preferred human monoclonal antibodies immunoreact with the CD4binding site of glycoprotein gp41. A preferred human monoclonal antibodyof this invention has the binding specificity of a monoclonal antibodycomprising a heavy chain immunoglobulin variable region amino acidresidue sequence selected from the group of sequences consisting of SEQID NOs 142, 143, 144, 145, and 146 and conservative substitutionsthereof.

Another preferred human monoclonal antibody of this invention has thegp41 binding specificity of a monoclonal antibody having a light chainimmunoglobulin variable region amino acid residue sequence selected fromthe group of sequences consisting of SEQ ID NOs 147, 148, 149, 150, and151 and conservative substitutions thereof.

As shown by the present teachings and using the combinatorial libraryshuffling and screening methods, one can identify new heavy and lightchain pairs that function as a HIV-neutralizing monoclonal antibody. Inparticular, one can shuffle a known heavy chain, derived from anHIV-neutralizing human monoclonal antibody, with a library of lightchains to identify new H:L pairs that form a functional antibodyaccording to the present invention. Similarly, one can shuffle a knownlight chain, derived from an HIV-neutralizing human monoclonal antibody,with a library of heavy chains to identify new H:L pairs that form afunctional antibody according to the present invention.

Particularly preferred human monoclonal antibodies are those having thegp120 immunoreaction (binding) specificity of a monoclonal antibodyhaving heavy and light chain immunoglobulin variable region amino acidresidue sequences in pairs (H:L) selected from the group consisting ofSEQ ID NOs 66:95, 67:96, 72:102, 66:97, 73:107, 74:103, 70:101, 68:98,75:104, 72:105, 78:110, 66:118, 66:122, 66:121, 66:115, 97:124, 97:132and 66:98, and conservative substitutions thereof. The designation oftwo SEQ ID NOs with a colon, e.g., 66:95, is to connote a H:L pairformed by the heavy and light chain, respectively, amino acid residuesequences shown in SEQ ID NO 66 and SEQ ID NO 95, respectively.

Further preferred human monoclonal antibodies are those having the gp41immunoreaction (binding) specificity of a monoclonal antibody havingheavy and light chain immunoglobulin variable region amino acid residuesequences in pairs (H:L) selected from the group consisting of SEQ IDNOs 142:147, 143:148, 144:149, 145:150, and 146:151, and conservativesubstitutions thereof.

Particularly preferred are human monoclonal antibodies having thebinding specificity of the monoclonal antibody produced by the E. colimicroorganisms deposited with the ATCC, as described further herein.

Particularly preferred are human monoclonal antibodies having thebinding specificity of the monoclonal antibodies produced by the E. colimicroorganisms designated ATCC 69078, 69079 and 69080. By "having thebinding specificity" is meant equivalent monoclonal antibodies whichexhibit the same or similar immunoreaction and neutralizationproperties, and which compete for binding to an HIV antigen. Preferredare the human monoclonal antibodies produced by ATCC 69078, 69079 and69080.

The term "conservative variation" as used herein denotes the replacementof an amino acid residue by another, biologically similar residue.Examples of conservative variations include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like. The term "conservativevariation" also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies having thesubstituted polypeptide also neutralize HIV. Analogously, anotherpreferred embodiment of the invention relates to polynucleotides whichencode the above noted heavy and/or light chain polypeptides and topolynucleotide sequences which are complementary to these polynucleotidesequences. Complementary polynucleotide sequences include thosesequences which hybridize to the polynucleotide sequences of theinvention under stringent hybridization conditions.

By using the human monoclonal antibodies of the invention, it is nowpossible to produce anti-idiotypic antibodies which can be used toscreen human monoclonal antibodies to identify whether the antibody hasthe same binding specificity as a human monoclonal antibody of theinvention and also used for active immunization (Herlyn et al., Science,232: 100 (1986)). Such anti-idiotypic antibodies can be produced usingwell-known hybridoma techniques (Kohler et al., Nature, 256: 495(1975)). An anti-idiotypic antibody is an antibody which recognizesunique determinants present on the human monoclonal antibody produced bythe cell line of interest. These determinants are located in thehypervariable region of the antibody. It is this region which binds to agiven epitope and, thus, is responsible for the specificity of theantibody. An anti-idiotypic antibody can be prepared by immunizing ananimal with the monoclonal antibody of interest. The immunized animalwill recognize and respond to the idiotypic determinants of theimmunizing antibody and produce an antibody to these idiotypicdeterminants. By using the anti-idiotypic antibodies of the immunizedanimal, which are specific for the human monoclonal antibody of theinvention produced by a cell line which was used to immunize the secondanimal, it is now possible to identify other clones with the sameidiotype as the antibody of the hybridoma used for immunization.Idiotypic identity between human monoclonal antibodies of two cell linesdemonstrates that the two monoclonal antibodies are the same withrespect to their recognition of the same epitopic determinant. Thus, byusing anti-idiotypic antibodies, it is possible to identify otherhybridomas expressing monoclonal antibodies having the same epitopicspecificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the"image" of the epitope bound by the first monoclonal antibody. Thus, theanti-idiotypic monoclonal antibody can be used for immunization, sincethe anti-idiotype monoclonal antibody binding domain effectively acts asan antigen.

In one preferred embodiment, the invention contemplates a truncatedimmunoglobulin molecule comprising a Fab fragment derived from a humanmonoclonal antibody of this invention. The Fab fragment, lacking Fcreceptor, is soluble, and affords therapeutic advantages in serum halflife, and diagnostic advantages in modes of using the soluble Fabfragment. The preparation of a soluble Fab fragment is generally knownin the immunological arts and can be accomplished by a variety ofmethods. A preferred method of producing a soluble Fab fragment isdescribed herein.

In another preferred embodiment, the invention contemplates animmunoglobulin molecule comprising a Fab fragment derived from a humanmonoclonal antibody of this invention and the fragment crystallizable(Fc) domain of a human immunoglobulin molecule. The entire (i.e.,complete) immunoglobulin (Ig) molecule comprising a Fab fragment withthe Fc domain may afford therapeutic and diagnostic advantages, and canbe any of the several Ig species depending upon the ultimate use,including IgG, IgA, IgD, IgE, IgM, and isotypes thereof. Theimmunoglobulin molecule would be capable of effector functionsassociated with the Fc domain when used in passive immunotherapy. Theseeffector functions include antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cellular cytotoxicity (CDCC) whichpromote the death of the cell to which the immunoglobulin molecule isspecifically bound. The effector functions may therefore be desirable intherapeutic applications. Diagnostic assays include the ability todetect the presence of the immunoglobulin molecule. These assays rely onthe cross-linking of red cells or beads in agglutinations, theactivation of complement in plaque assays, or the antigenic propertiesof the Fc region of the heavy chain as detected by secondary antibodiesin ELISA or RIA procedures to detect the presence of the immunoglobulinmolecule. Such diagnostic assays can only be performed with the entireimmunoglobulin molecule. The isolation of the immunoglobulin molecule isalso facilitated by the presence of the Fc domain in that commonly usedmethods of immunoglobulin purification are based upon interaction ofreagents with the Fc domain. The preparation of a Fab fragment with theFc domain is generally known in the immunological arts and can beaccomplished by a variety of methods. A preferred method of producing aFab fragment with the Fc domain is described herein.

Particularly preferred is the immunoglobulin IgG1 human antibodydescribed herein that is comprised of the b12 antibody Fab fragment andhuman Fc domain derived from an IgG1 subtype, designated b12 IgG1. Thestructure and preparation of this preferred human monoclonal antibody isdescribed herein, and is prepared using the recombinant DNA expressionvector pEE12. The nucleotide sequences for preferred heavy and lightchains are also shown in SEQ ID NOs 169 and 168, respectively. Thecomplete nucleotide sequence of the vector for expression the completeheavy and light chains in the form of b12 IgG1 is shown in FIG. 27 andalso in SEQ ID NOs 156 and 170. Accordingly, the amino acid residue andnucleotide sequences, respectively, for a preferred complete heavy chainare shown in SEQ ID NOs 155 and 154, respectively, and for a preferredlight chain are shown in SEQ ID NOs 153, and 152, respectively.

C. Immunotherapeutic Methods and Compositions

The human monoclonal antibodies can also be used immunotherapeuticallyfor HIV disease. The term "immunotherapeutically" or "immunotherapy" asused herein in conjunction with the monoclonal antibodies of theinvention denotes both prophylactic as well as therapeuticadministration. Thus, the monoclonal antibodies can be administered tohigh-risk patients in order to lessen the likelihood and/or severity ofHIV-induced disease, administered to patients already evidencing activeHIV infection, or administered to patients at risk of HIV infection.

1. Therapeutic Compositions

The present invention therefore contemplates therapeutic compositionsuseful for practicing the therapeutic methods described herein.Therapeutic compositions of the present invention contain aphysiologically tolerable carrier together with at least one species ofhuman monoclonal antibody as described herein, dissolved or dispersedtherein as an active ingredient. In a preferred embodiment, thetherapeutic composition is not immunogenic when administered to a humanpatient for therapeutic purposes, unless that purpose is to induce animmune response, as described elsewhere herein.

As used herein, the terms "pharmaceutically acceptable","physiologically tolerable" and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a human without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in theart. Typically such compositions are prepared as sterile injectableseither as liquid solutions or suspensions, aqueous or non-aqueous,however, solid forms suitable for solution, or suspensions, in liquidprior to use can also be prepared. The preparation can also beemulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, propylene glycol,polyethylene glycol and other solutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, organic esters such asethyl oleate, and water-oil emulsions.

A therapeutic composition contains an HIV-neutralizing of a humanmonoclonal antibody of the present invention, typically an amount of atleast 0.1 weight percent of antibody per weight of total therapeuticcomposition. A weight percent is a ratio by weight of antibody to totalcomposition. Thus, for example, 0.1 weight percent is 0.1 grams ofantibody per 100 grams of total composition.

2. Therapeutic Methods

In view of the demonstrated HIV neutralizing ability of the humanmonoclonal antibodies of the present invention, the present disclosureprovides for a method for neutralizing HIV in vitro or in vivo. Themethod comprises contacting a sample believed to contain HIV with acomposition comprising a therapeutically effective amount of a humanmonoclonal antibody of this invention.

For in vivo modalities, the method comprises administering to thepatient a therapeutically effective amount of a physiologicallytolerable composition containing a human monoclonal antibody of theinvention. Thus, the present invention describes in one embodiment amethod for providing passive immunotherapy to HIV disease in a humancomprising administering to the human an immunotherapeutically effectiveamount of the monoclonal antibody of this invention.

A representative patient for practicing the present passiveimmunotherapeutic methods is any human exhibiting symptoms ofHIV-induced disease, including AIDS or related conditions believed to becaused by HIV infection, and humans at risk of HIV infection. Patientsat risk of infection by HIV include babies of HIV-infected pregnantmothers, recipients of transfusions known to contain HIV, users of HIVcontaminated needles, individuals who have participated in high risksexual activities with known HIV-infected individuals, and the like risksituations.

In one embodiment, the passive immunization method comprisesadministering a composition comprising more than one species of humanmonoclonal antibody of this invention, preferably directed tonon-competing epitopes or directed to distinct serotypes or strains ofHIV, as to afford increased effectiveness of the passive immunotherapy.

A therapeutically (immunotherapeutically) effective amount of a humanmonoclonal antibody is a predetermined amount calculated to achieve thedesired effect, i.e., to neutralize the HIV present in the sample or inthe patient, and thereby decrease the amount of detectable HIV in thesample or patient. In the case of in vivo therapies, an effective amountcan be measured by improvements in one or more symptoms associated withHIV-induced disease occurring in the patient, or by serologicaldecreases in HIV antigens.

Thus, the dosage ranges for the administration of the monoclonalantibodies of the invention are those large enough to produce thedesired effect in which the symptoms of the HIV disease are amelioratedor the likelihood of infection decreased. The dosage should not be solarge as to cause adverse side effects, such as hyperviscositysyndromes, pulmonary edema, congestive heart failure, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient and can be determined by one of skill inthe art.

The dosage can be adjusted by the individual physician in the event ofany complication.

A therapeutically effective amount of an antibody of this invention istypically an amount of antibody such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.1 microgram (ug) per milliliter (ml) toabout 100 ug/ml, preferably from about 1 ug/ml to about 5 ug/ml, andusually about 5 ug/ml. Stated differently, the dosage can vary fromabout 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg toabout 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg,in one or more dose administrations daily, for one or several days.

The human monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual infusion over time. Although theHIV infection is typically systemic and therefore most often treated byintravenous administration of therapeutic compositions, other tissuesand delivery means are contemplated where there is a likelihood that thetissue targeted contains infectious HIV. Thus, human monoclonalantibodies of the invention can be administered intravenously,intraperitoneally, intramuscularly, subcutaneously, intracavity,transdermally, and can be delivered by peristaltic means.

The therapeutic compositions containing a human monoclonal antibody ofthis invention are conventionally administered intravenously, as byinjection of a unit dose, for example. The term "unit dose" when used inreference to a therapeutic composition of the present invention refersto physically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgement of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for administration are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

As an aid to the administration of effective amounts of a monoclonalantibody, a diagnostic method for detecting a monoclonal antibody in thesubject's blood is useful to characterize the fate of the administeredtherapeutic composition.

The invention also relates to a method for preparing a medicament orpharmaceutical composition comprising the human monoclonal antibodies ofthe invention, the medicament being used for immunotherapy of HIVdisease.

D. Diagnostic Assay Methods

The present invention contemplates various assay methods for determiningthe presence, and preferably amount, of HIV in a sample such as abiological fluid or tissue sample using a human monoclonal antibody ofthis invention as an immunochemical reagent to form an immunoreactionproduct whose amount relates, either directly or indirectly, to theamount of HIV in the sample.

In a related embodiment, the present invention contemplates variousassay methods for determining the presence, and preferably amount, of ananti-HIV antibody present in a sample such as a biological fluid ortissue sample from a HIV-infected individual using a human monoclonalantibody of this invention as an immunochemical reagent to form animmunoreaction product whose amount relates, either directly orindirectly, to the amount of anti-HIV antibody in the sample.

Those skilled in the art will understand that there are numerous wellknown clinical diagnostic chemistry procedures in which animmunochemical reagent of this invention can be used to form animmunoreaction product whose amount relates to the amount of HIV oranti-HIV antibody present in a body sample. Thus, while exemplary assaymethods are described herein, the invention is not so limited.

Various heterogenous and homogeneous protocols, either competitive ornoncompetitive, can be employed in performing an assay method of thisinvention. Examples of types of immunoassays which can utilizemonoclonal antibodies of the invention are competitive andnon-competitive immunoassays in either a direct or indirect format.Examples of such immunoassays are the radioimmunoassay (RIA) and thesandwich (immunometric) assay.

Detection of the antigens using the monoclonal antibodies of theinvention can be done utilizing immunoassays which are run in either theforward, reverse, or simultaneous modes, including immunohistochemicalassays on physiological samples. Those of skill in the art will know, orcan readily discern, other immunoassay formats without undueexperimentation.

The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of HIV. Examples ofwell-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such, using routineexperimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds, andbio-luminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for binding to the monoclonal antibodies of theinvention, or will be able to ascertain such, using routineexperimentation. Furthermore, the binding of these labels to themonoclonal antibodies of the invention can be done using standardtechniques common to those of ordinary skill in the art.

For purposes of the invention, HIV may be detected by the monoclonalantibodies of the invention when present in samples of biological fluidsand tissues. Any sample containing a detectable amount of HIV can beused. A sample can be a liquid such as urine, saliva, cerebrospinalfluid, blood, serum and the like, or a solid or semi-solid such astissues, feces, and the like, or, alternatively, a solid tissue such asthose commonly used in histological diagnosis.

Another labeling technique which may result in greater sensitivityconsists of coupling the antibodies to low molecular weight haptens.These haptens can then be specifically detected by means of a secondreaction. For example, it is common to use haptens such as biotin, whichreacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, whichcan react with specific anti-hapten antibodies.

The monoclonal antibodies of the invention are suited for use in vitro,for example, in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier for the detection of HIV insamples, as described above. The monoclonal antibodies in theseimmunoassays can be detectably labeled in various ways for in vitro use.

In using the human monoclonal antibodies of the invention for the invivo detection of antigen, the detectably labeled human monoclonalantibody is given in a dose which is diagnostically effective. The term"diagnostically effective" means that the amount of detectably labeledhuman monoclonal antibody is administered in sufficient quantity toenable detection of the site having the HIV antigen for which themonoclonal antibodies are specific.

The concentration of detectably labeled human monoclonal antibody whichis administered should be sufficient such that the binding to HIV isdetectable compared to the background. Further, it is desirable that thedetectably labeled monoclonal antibody be rapidly cleared from thecirculatory system in order to give the best target-to-background signalratio.

As a rule, the dosage of detectably labeled human monoclonal antibodyfor in vivo diagnosis will vary depending on such factors as age, sex,and extent of disease of the individual. The dosage of human monoclonalantibody can vary from about 0.01 mg/m² to about 500 mg/m², preferably0.1 mg/m² to about 200 mg/m², most preferably about 0.1 mg/m² to about10 mg/m². Such dosages may vary, for example, depending on whethermultiple injections are given, tissue, and other factors known to thoseof skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹ In, ⁹⁷ Ru, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² AS, ⁸⁹Zr, and ²⁰¹ Tl.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵²Cr, and ⁵⁶ Fe.

The human monoclonal antibodies of the invention can be used in vitroand in vivo to monitor the course of HIV disease therapy. Thus, forexample, by measuring the increase or decrease in the number of cellsinfected with HIV or changes in the concentration of HIV present in thebody or in various body fluids, it would be possible to determinewhether a particular therapeutic regimen aimed at ameliorating the HIVdisease is effective.

In a related diagnostic embodiment, the invention contemplates screeningHIV-infected patients for the presence of circulating anti-HIVantibodies immunoreactive with gp120 that have a similar epitopeimmunospecificity when compared to a neutralizing antibody of thisinvention. Such a screening method indicates that the HIV-infectedpatient is exhibiting a significant immune response to the virus, andprovides useful information regarding disease status and prognosis. Thepresence of anti-HIV antibodies cross-reactive with a neutralizingantibody of this invention indicates that the patient has some degree ofHIV neutralizing activity, as defined herein.

The diagnostic assay involves determining whether the patient containshuman anti-HIV antibodies immunoreactive with the same, similar oroverlapping epitopes as a neutralizing antibody of the invention, suchthat there is a likelihood that there is a useful neutralizing immuneresponse in the patient. There are a variety of immunological assayformats that can be utilized to determine cross-reactivity of test andcontrol antibodies, and the invention need not be so limiting.Particularly preferred are competition assays for a common antigen,preferably in the solid phase.

A preferred embodiment of the competition immunoassay method comprisesthe steps of:

(1) contacting a sample believed to contain a human anti-HIV antibodywith a diagnostically effective amount of the monoclonal antibodydescribed herein that binds mature gp120 in a competition immunoreactionadmixture containing mature gp120 in the solid phase;

(2) maintaining said competition immunoreaction admixture underconditions sufficient for said monoclonal antibody to bind with saidgp120 in the solid phase and form a solid phase immunoreactant; and

(3) detecting the amount of said immunoreactant present in said solidphase, and thereby the immunocompetence of any human anti-HIV antibodyin said sample.

A diagnostically effective amount, in this context, is a amount relativeto the solid phase gp120, preferably "mature" gp120 as defined herein,sufficient to produce a detectable solid phase immunoreaction productbetween the solid phase gp120 and the control anti-gp120 antibody ofthis invention. Exemplary competition assays are described herein usingthe preferred b12 antibody.

Conditions for conducting the competition immunoreaction are well knownin the art and can be varied according to recognized parameters in thecontacting, the reaction admixtures, the maintenance step, theimmunoreaction conditions and the detecting step. For example, thedetection step can be conducted by use of a labeled antibody of thisinvention, by use of a second, labeled anti-human antibody, and thelike, as described herein.

E. Diagnostic Systems

The present invention also describes a diagnostic system, preferably inkit form, for assaying for the presence of HIV or an anti-HIV antibodyin a sample according to the diagnostic methods described herein. Adiagnostic system includes, in an amount sufficient to perform at leastone assay, a subject human monoclonal antibody, as a separately packagedreagent.

In another embodiment, a diagnostic system is contemplated for assayingfor the presence of an anti-HIV monoclonal antibody in a body fluidsample such as for monitoring the fate of therapeutically administeredantibody. The system includes, in an amount sufficient for at least oneassay, a subject antibody as a control reagent, and preferably apreselected amount of HIV antigen, each as separately packagedimmunochemical reagents.

Instructions, for use of the packaged reagent are also typicallyincluded.

"Instructions for use" typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

In embodiments for detecting HIV or anti-HIV antibody in a body fluid, adiagnostic system of the present invention can include a label orindicating means capable of signaling the formation of an immunocomplexcontaining a human monoclonal antibody of the present invention.

The word "complex" as used herein refers to the product of a specificbinding reaction such as an antibody-antigen reaction. Exemplarycomplexes are immunoreaction products.

As used herein, the terms "label" and "indicating means" in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. Any label or indicatingmeans can be linked to or incorporated in an expressed protein,polypeptide, or antibody molecule that is part of an antibody ormonoclonal antibody composition of the present invention, or usedseparately, and those atoms or molecules can be used alone or inconjunction with additional reagents. Such labels are themselveswell-known in clinical diagnostic chemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelproteins methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturing them to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyanate (FITC),5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca,"Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such ashorseradish peroxidase glucose oxidase, or the like. In such cases wherethe principal indicating group is an enzyme such as HRP or glucoseoxidase, additional reagents are required to visualize the fact that areceptor-ligand complex (immunoreactant) has formed. Such additionalreagents for HRP include hydrogen peroxide and an oxidation dyeprecursor such as diaminobenzidine. An additional reagent useful withglucose oxidase is 2,2'-amino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

Radioactive elements are also useful labeling agents and are usedillustratively herein. An exemplary radiolabeling agent is a radioactiveelement that produces gamma ray emissions. Elements which themselvesemit gamma rays, such as ¹²⁴ I, ¹²⁵ I, ¹²⁸ I, ¹³² I and ⁵¹ Cr representone class of gamma ray emission-producing radioactive element indicatinggroups. Particularly preferred is ¹²⁵ I. Another group of usefullabeling means are those elements such as ¹¹ C, ¹⁸ F, ¹⁵ O and ¹³ Nwhich themselves emit positrons. The positrons so emitted produce gammarays upon encounters with electrons present in the animal's body. Alsouseful is a beta emitter, such ¹¹¹ indium of ³ H.

The linking of labels, i.e., labeling of, polypeptides and proteins iswell known in the art. For instance, antibody molecules produced by ahybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable. See, forexample, Aurameas et al., Scand. J. Immunol., Vol. 8 Suppl. 7: 7-23(1978), Rodwell et al., Biotech., 3: 889-894 (1984), and U.S. Pat. No.4,493,795.

The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A "specific binding agent" is amolecular entity capable of selectively binding a reagent species of thepresent invention or a complex containing such a species, but is notitself a polypeptide or antibody molecule composition of the presentinvention. Exemplary specific binding agents are second antibodymolecules, complement proteins or fragments thereof, S. aureus proteinA, and the like. Preferably the specific binding agent binds the reagentspecies when that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled Specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an "ELISA"format to detect the quantity of an antigen or antibody of thisinvention in a vascular fluid sample such as blood, serum, or plasma."ELISA" refers to an enzyme-linked immunosorbent assay that employs anantibody or antigen bound to a solid phase and an enzyme-antigen orenzyme-antibody conjugate to detect and quantify the amount of anantigen present in a sample. A description of the ELISA technique isfound in Chapter 22 of the 4th Edition of Basic and Clinical Immunologyby D. P. Sites et al., published by Lange Medical Publications of LosAltos, Calif. in 1982 and in U.S. Pat. Nos. 3,654,090; 3,850,752; and4,016,043, which are all incorporated herein by reference.

Thus, in some embodiments, a human monoclonal antibody of the presentinvention can be affixed to a solid matrix to form a solid support thatcomprises a package in the subject diagnostic systems.

A reagent is typically affixed to a solid matrix by adsorption from anaqueous medium although other modes of affixation applicable to proteinsand polypeptides well known to those skilled in the art, can be used.

Useful solid matrices are also well known in the art. Such materials arewater insoluble and include the cross-linked dextran available under thetrademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.);agarose; beads of polystyrene beads about 1 micron to about 5millimeters in diameter available from Abbott Laboratories of NorthChicago, Ill.; polyvinyl chloride, polystyrene, cross-linkedpolyacrylamide, nitrocellulose- or nylon-based webs such as sheets,strips or paddles; or tubes, plates or the wells of a microtiter platesuch as those made from polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microtiter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnosticsystems are those customarily utilized in diagnostic systems.

The term "package" refers to a solid matrix or material such as glass,plastic (e.g., polyethylene, polypropylene and polycarbonate), paper,foil and the like capable of holding within fixed limits a diagnosticreagent such as a monoclonal antibody of the present invention. Thus,for example, a package can be a bottle, vial, plastic and plastic-foillaminated envelope or the like container used to contain a contemplateddiagnostic reagent or it can be a microtiter plate well to whichmicrogram quantities of a contemplated diagnostic reagent have beenoperatively affixed, i.e., linked so as to be capable of beingimmunologically bound by an antibody or polypeptide to be detected.

The materials for use in the assay of the invention are ideally suitedfor the preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the container means may comprise a humanmonoclonal antibody of the invention which is, or can be, detectablylabelled. The kit may also have containers containing any of the otherabove-recited immunochemical reagents used to practice the diagnosticmethods.

F. Methods for Producing an HIV-Neutralizing Human Monoclonal Antibody

The present invention describes methods for producing novelHIV-neutralizing human monoclonal antibodies. The methods are basedgenerally on the use of combinatorial libraries of antibody moleculeswhich can be produced from a variety of sources, and include naivelibraries, modified libraries, and libraries produced directly fromhuman donors exhibiting an HIV-specific immune response.

The combinatorial library production and manipulation methods have beenextensively described in the literature, and will not be reviewed indetail herein, except for those feature required to make and use uniqueembodiments of the present invention. However, the methods generallyinvolve the use of a filamentous phage (phagemid) surface expressionvector system for cloning and expressing antibody species of thelibrary. Various phagemid cloning systems to produce combinatoriallibraries have been described by others. See, for example thepreparation of combinatorial antibody libraries on phagemids asdescribed by Kang et al., Proc. Natl. Acad. Sci., USA, 88: 4363-4366(1991); Barbas et al., Proc. Natl. Acad. Sci., USA, 88: 7978-7982(1991); Zebedee et al., Proc. Natl. Acad. Sci., USA, 89: 3175-3179(1992); Kang et al., Proc. Natl. Acad. Sci., USA, 88: 11120-11123(1991); Barbas et al., Proc. Natl. Acad. Sci., USA, 89: 4457-4461(1992); and Gram et al., Proc. Natl. Acad. Sci., USA, 89: 3576-3580(1992), which references are hereby incorporated by reference.

In one embodiment, the method involves preparing a phagemid library ofhuman monoclonal antibodies by using donor immune cell messenger RNAfrom HIV-infected donors. The donors can be symptomatic of AIDS, but inpreferred embodiments the donor is asymptomatic, as the resultinglibrary contains a substantially higher number of HIV-neutralizing humanmonoclonal antibodies.

In another embodiment, the donor is naive relative to an immune responseto HIV, i.e., the donor is not HIV-infected. Alternatively, the librarycan be synthetic, or can be derived from a donor who has an immuneresponse to other antigens.

The method for producing a human monoclonal antibody generally involves(1) preparing separate H and L chain-encoding gene libraries in cloningvectors using human immunoglobulin genes as a source for the libraries,(2) combining the H and L chain encoding gene libraries into a singledicistronic expression vector capable of expressing and assembling aheterodimeric antibody molecule, (3) expressing the assembledheterodimeric antibody molecule on the surface of a filamentous phageparticle, (4) isolating the surface-expressed phage particle usingimmunoaffinity techniques such as panning of phage particles against apreselected antigen, thereby isolating one or more species of phagemidcontaining particular H and L chain-encoding genes and antibodymolecules that immunoreact with the preselected antigen.

As described herein the Examples, the resulting phagemid library can bemanipulated to increase and/or alter the immunospecificities of themonoclonal antibodies of the library to produce and subsequentlyidentify additional, desirable, human monoclonal antibodies of thepresent invention.

For example, the heavy (H) chain and light (L) chain immunoglobulinmolecule encoding genes can be randomly mixed (shuffled) to create newHL pairs in an assembled immunoglobulin molecule. Additionally, eitheror both the H and L chain encoding genes can be mutagenized in thecomplementarity determining region (CDR) of the variable region of theimmunoglobulin polypeptide, and subsequently screened for desirableimmunoreaction and neutralization capabilities.

In one embodiment, the H and L genes can be cloned into separate,monocistronic expression vectors, referred to as a "binary" systemdescribed further herein. In this method, step (2) above differs in thatthe combining of H and L chain encoding genes occurs by theco-introduction of the two binary plasmids into a single host cell forexpression and assembly of a phagemid having the surface accessibleantibody heterodimer molecule.

In one shuffling embodiment, the shuffling can be accomplished with thebinary expression vectors, each capable of expressing a single heavy orlight chain encoding gene, as described in Example 11.

In the present methods, the antibody molecules are monoclonal becausethe cloning methods allow for the preparation of clonally pure speciesof antibody producing cell lines. In addition, the monoclonal antibodiesare human because the H and L chain encoding genes are derived fromhuman immunoglobulin producing immune cells, such as spleen, thymus,bone marrow, and the like.

The method of producing a HIV-neutralizing human monoclonal antibodyalso requires that the resulting antibody library, immunoreactive with apreselected HIV antigen, is screened for the presence of antibodyspecies which have the capacity to neutralize HIV in one or more of theassays described herein for determining neutralization capacity. Thus, apreferred library of antibody molecules is first produced which binds toan HIV antigen, preferably gp160, gp120, gp41, the V3 loop region ofgp160, or the CD4 binding site of gp120 and gp41, and then is screenedfor the presence of HIV-neutralizing antibodies as described herein.

Additional libraries can be screened from shuffled libraries foradditional HIV-immunoreactive and neutralizing human monoclonalantibodies.

As a further characterization of the present invention the nucleotideand corresponding amino acid residue sequence of the antibody molecule'sH or L chain encoding gene is determined by nucleic acid sequencing. Theprimary amino acid residue sequence information provides essentialinformation regarding the antibody molecule's epitope reactivity.

Sequence comparisons of identified HIV-immunoreactive monoclonalantibody variable chain region sequences are shown herein in FIGS.10-13. The sequences are aligned based on sequence homology, and groupsof related antibody molecules are identified thereby in which heavychain or light chain genes share substantial sequence homology.

An exemplary preparation of a human monoclonal antibody is described inthe Examples. The isolation of a particular vector capable of expressingan antibody of interest involves the introduction of the dicistronicexpression vector into a host cell permissive for expression offilamentous phage genes and the assembly of phage particles. Where thebinary vector system is used, both vectors are introduced in the hostcell. Typically, the host is E. coli. Thereafter, a helper phage genomeis introduced into the host cell containing the immunoglobulinexpression vector(s) to provide the genetic complementation necessary toallow phage particles to be assembled. The resulting host cell iscultured to allow the introduced phage genes and immunoglobulin genes tobe expressed, and for phage particles to be assembled and shed from thehost cell. The shed phage particles are then harvested (collected) fromthe host cell culture media and screened for desirable immunoreactionand neutralization properties. Typically, the harvested particles are"panned" for immunoreaction with a preselected antigen. The stronglyimmunoreactive particles are then collected, and individual species ofparticles are clonally isolated and further screened for HIVneutralization. Phage which produce neutralizing antibodies are selectedand used as a source of a human HIV neutralizing monoclonal antibody ofthis invention.

Human monoclonal antibodies of this invention can also be produced byaltering the nucleotide sequence of a polynucleotide sequence thatencodes a heavy or light chain of a monoclonal antibody of thisinvention. For example, by site directed mutagenesis, one can alter thenucleotide sequence of an expression vector and thereby introducechanges in the resulting expressed amino acid residue sequence. Thus onecan take the polynucleotide of SEQ ID NO 66, for example, and convert itinto the polynucleotide of SEQ ID NO 67. Similarly, one can take a knownpolynucleotide and randomly alter it by random mutagenesis, reintroducethe altered polynucleotide into an expression system and subsequentlyscreen the product H:L pair for HIV-neutralizing activity.

Site-directed and random mutagenesis methods are well known in thepolynucleotide arts, and are not to be construed as limiting as methodsfor altering the nucleotide sequence of a subject polynucleotide.

Due to the presence of the phage particle in an immunoaffinity isolatedantibody, one embodiment involves the manipulation of the resultingcloned genes to truncate the immunoglobulin-coding gene such that asoluble Fab fragment is secreted by the host E. coli cell containing thephagemid vector. Thus, the resulting manipulated cloned immunoglobulingenes produce a soluble Fab which can be readily characterized in ELISAassays for epitope binding studies, in competition assays with knownanti-HIV antibody molecules, and in HIV neutralization assays. Thesolubilized Fab provides a reproducible and comparable antibodypreparation for comparative and characterization studies.

The preparation of soluble Fab is generally described in theimmunological arts, and can be conducted as described herein in Example2b6), or as described by Burton et al., Proc. Natl. Acad. Sci., USA, 88:10134-10137 (1991).

G. Expression Vectors and Polynucleotides for Expressing Anti-HIVMonoclonal Antibodies

The preparation of human monoclonal antibodies of this inventiondepends, in one embodiment, on the cloning and expression vectors usedto prepare the combinatorial antibody libraries described herein. Thecloned immunoglobulin heavy and light chain genes can be shuttledbetween lambda vectors, phagemid vectors and plasmid vectors at variousstages of the methods described herein.

The phagemid vectors produce fusion proteins that are expressed on thesurface of an assembled filamentous phage particle.

A preferred phagemid vector of the present invention is a recombinantDNA (rDNA) molecule containing a nucleotide sequence that codes for andis capable of expressing a fusion polypeptide containing, in thedirection of amino- to carboxy-terminus, (1) a prokaryotic secretionsignal domain, (2) a heterologous polypeptide defining an immunoglobulinheavy or light chain variable region, and (3) a filamentous phagemembrane anchor domain. The vector includes DNA expression controlsequences for expressing the fusion polypeptide, preferably prokaryoticcontrol sequences.

The filamentous phage membrane anchor is preferably a domain of thecpIII or cpVIII coat protein capable of associating with the matrix of afilamentous phage particle, thereby incorporating the fusion polypeptideonto the phage surface.

The secretion signal is a leader peptide domain of a protein thattargets the protein to the periplasmic membrane of gram negativebacteria. A preferred secretion signal is a pelB secretion signal. Thepredicted amino acid residue sequences of the secretion signal domainfrom two pelB gene product variants from Erwinia carotova are describedin Lei et al., Nature, 331: 543-546 (1988).

The leader sequence of the pelB protein has previously been used as asecretion signal for fusion proteins (Better et al., Science, 240:1041-1043 (1988); Sastry et al., Proc. Natl. Acad. Sci., USA, 86:5728-5732 (1989); and Mullinax et al., Proc. Natl. Acad. Sci., USA, 87:8095-8099 (1990)). Amino acid residue sequences for other secretionsignal polypeptide domains from E. coli useful in this invention asdescribed in Oliver, Escherichia coli and Salmonella Typhimurium,Neidhard, F. C. (ed.), American Society for Microbiology, Washington,D.C., 1: 56-69 (1987).

Preferred membrane anchors for the vector are obtainable fromfilamentous phage M13, f1, fd, and equivalent filamentous phage.Preferred membrane anchor domains are found in the coat proteins encodedby gene III and gene VIII. The membrane anchor domain of a filamentousphage coat protein is a portion of the carboxy terminal region of thecoat protein and includes a region of hydrophobic amino acid residuesfor spanning a lipid bilayer membrane, and a region of charged aminoacid residues normally found at the cytoplasmic face of the membrane andextending away from the membrane.

In the phage f1, gene VIII coat protein's membrane spanning regioncomprises residue Trp-26 through Lys-40, and the cytoplasmic regioncomprises the carboxy-terminal 11 residues from 41 to 52 (Ohkawa et al.,J. Biol. Chem., 256: 9951-9958 (1981)). An exemplary membrane anchorwould consist of residues 26 to 40 of cpVIII. Thus, the amino acidresidue sequence of a preferred membrane anchor domain is derived fromthe M13 filamentous phage gene VIII coat protein (also designated cpVIIIor CP 8). Gene VIII coat protein is present on a mature filamentousphage over the majority of the phage particle with typically about 2500to 3000 copies of the coat protein.

In addition, the amino acid residue sequence of another preferredmembrane anchor domain is derived from the M13 filamentous phage geneIII coat protein (also designated cpIII). Gene III coat protein ispresent on a mature filamentous phage at one end of the phage particlewith typically about 4 to 6 copies of the coat protein.

For detailed descriptions of the structure of filamentous phageparticles, their coat proteins and particle assembly, see the reviews byRached et al., Microbiol. Rev., 50: 401-427 (1986); and Model et al., in"The Bacteriophages: Vol. 2", R. Calendar, ed. Plenum Publishing Co.,pp. 375-456 (1988).

DNA expression control sequences comprise a set of DNA expressionsignals for expressing a structural gene product and include both 5' and3' elements, as is well known, operatively linked to the cistron suchthat the cistron is able to express a structural gene product. The 5'control sequences define a promoter for initiating transcription and aribosome binding site operatively linked at the 5' terminus of theupstream translatable DNA sequence.

To achieve high levels of gene expression in E. coli, it is necessary touse not only strong promoters to generate large quantities of mRNA, butalso ribosome binding sites to ensure that the mRNA is efficientlytranslated. In E. coli, the ribosome binding site includes an initiationcodon (AUG) and a sequence 3-9 nucleotides long located 3-11 nucleotidesupstream from the initiation codon (Shine et al., Nature, 254: 34(1975). The sequence, AGGAGGU, which is called the Shine-Dalgarno (SD)sequence, is complementary to the 3' end of E. coli 16S rRNA. Binding ofthe ribosome to mRNA and the sequence at the 3' end of the mRNA can beaffected by several factors:

(i) The degree of complementarity between the SD sequence and 3' end ofthe 16S rRNA.

(ii) The spacing and possibly the DNA sequence lying between the SDsequence and the AUG. Roberts et al., Proc. Natl. Acad. Sci., USA, 76:760, (1979a); Roberts et al., Proc. Natl. Acad. Sci. USA, 76: 5596(1979b); Guarente et al., Science, 209: 1428 (1980); and Guarente etal., Cell, 20: 543 (1980). Optimization is achieved by measuring thelevel of expression of genes in plasmids in which this spacing issystematically altered. Comparison of different mRNAs shows that thereare statistically preferred sequences from positions -20 to +13 (wherethe A of the AUG is position 0). Gold et al., Annu. Rev. Microbiol., 35:365 (1981). Leader sequences have been shown to influence translationdramatically. Roberts et al., 1979 a, b supra.

(iii) The nucleotide sequence following the AUG, which affects ribosomebinding. Taniguchi et al., J. Mol. Biol., 118: 533 (1978).

The 3' control sequences define at least one termination (stop) codon inframe with and operatively linked to the heterologous fusionpolypeptide.

In preferred embodiments, the vector utilized includes a prokaryoticorigin of replication or replicon, i.e., a DNA sequence having theability to direct autonomous replication and maintenance of therecombinant DNA molecule extra chromosomally in a prokaryotic host cell,such as a bacterial host cell, transformed therewith. Such origins ofreplication are well known in the art. Preferred origins of replicationare those that are efficient in the host organism. A preferred host cellis E. coli. For use of a vector in E. coli, a preferred origin ofreplication is ColE1 found in pBR322 and a variety of other commonplasmids. Also preferred is the p15A origin of replication found onpACYC and its derivatives. The ColE1 and p15A replicon have beenextensively utilized in molecular biology, are available on a variety ofplasmids and are described at least by Sambrook et al., in "MolecularCloning: a Laboratory Manual", 2nd edition, Cold Spring HarborLaboratory Press (1989).

The ColE1 and p15A replicons are particularly preferred for use in oneembodiment of the present invention where two "binary" plasmids areutilized because they each have the ability to direct the replication ofplasmid in E. coli while the other replicon is present in a secondplasmid in the same E. coli cell. In other words, ColE1 and p15A arenon-interfering replicons that allow the maintenance of two plasmids inthe same host (see, for example, Sambrook et al., supra, at pages1.3-1.4). This feature is particularly important in the binary vectorsembodiment of the present invention because a single host cellpermissive for phage replication must support the independent andsimultaneous replication of two separate vectors, namely a first vectorfor expressing a heavy chain polypeptide, and a second vector forexpressing a light chain polypeptide.

In addition, those embodiments that include a prokaryotic replicon canalso include a gene whose expression confers a selective advantage, suchas drug resistance, to a bacterial host transformed therewith. Typicalbacterial drug resistance genes are those that confer resistance toampicillin, tetracycline, neomycin/kanamycin or cholamphenicol. Vectorstypically also contain convenient restriction sites for insertion oftranslatable DNA sequences. Exemplary vectors are the plasmids pUCS,pUC9, pBR322, and pBR329 available from BioRad Laboratories, (Richmond,Calif.) and pPL and pKK223 available from Pharmacia, (Piscataway, N.J.).

A vector for expression of a monoclonal antibody of the invention on thesurface of a filamentous phage particle is a recombinant DNA (rDNA)molecule adapted for receiving and expressing translatable first andsecond DNA sequences in the form of first and second polypeptideswherein one of the polypeptides is fused to a filamentous phage coatprotein membrane anchor. That is, at least one of the polypeptides is afusion polypeptide containing a filamentous phage membrane anchordomain, a prokaryotic secretion signal domain, and an immunoglobulinheavy or light chain variable domain.

A DNA expression vector for expressing a heterodimeric antibody moleculeprovides a system for independently cloning (inserting) the twotranslatable DNA sequences into two separate cassettes present in thevector, to form two separate cistrons for expressing the first andsecond polypeptides of the antibody molecule, or the ligand bindingportions of the polypeptides that comprise the antibody molecule (i.e.,the H and L variable regions of an immunoglobulin molecule). The DNAexpression vector for expressing two cistrons is referred to as adicistronic expression vector.

The vector comprises a first cassette that includes upstream anddownstream translatable DNA sequences operatively linked via a sequenceof nucleotides adapted for directional ligation to an insert DNA. Theupstream translatable sequence encodes the secretion signal as definedherein. The downstream translatable sequence encodes the filamentousphage membrane anchor as defined herein. The cassette preferablyincludes DNA expression control sequences for expressing the receptorpolypeptide that is produced when an insert translatable DNA sequence(insert DNA) is directionally inserted into the cassette via thesequence of nucleotides adapted for directional ligation. Thefilamentous phage membrane anchor is preferably a domain of the cpIII orcpVIII coat protein capable of binding the matrix of a filamentous phageparticle, thereby incorporating the fusion polypeptide onto the phagesurface.

The receptor expressing vector also contains a second cassette forexpressing a second receptor polypeptide. The second cassette includes asecond translatable DNA sequence that encodes a secretion signal, asdefined herein, operatively linked at its 3' terminus via a sequence ofnucleotides adapted for directional ligation to a downstream DNAsequence of the vector that typically defines at least one stop codon inthe reading frame of the cassette. The second translatable DNA sequenceis operatively linked at its 5' terminus to DNA expression controlsequences forming the 5' elements. The second cassette is capable, uponinsertion of a translatable DNA sequence (insert DNA), of expressing thesecond fusion polypeptide comprising a receptor of the secretion signalwith a polypeptide coded by the insert DNA.

An upstream translatable DNA sequence encodes a prokaryotic secretionsignal as described earlier. The upstream translatable DNA sequenceencoding the pelB secretion signal is a preferred DNA sequence forinclusion in a receptor expression vector. A downstream translatable DNAsequence encodes a filamentous phage membrane anchor as describedearlier. Thus, a downstream translatable DNA sequence encodes an aminoacid residue sequence that corresponds, and preferably is identical, tothe membrane anchor domain of either a filamentous phage gene III orgene VIII coat polypeptide.

A cassette in a DNA expression vector of this invention is the region ofthe vector that forms, upon insertion of a translatable DNA sequence(insert DNA), a sequence of nucleotides capable of expressing, in anappropriate host, a fusion polypeptide. The expression-competentsequence of nucleotides is referred to as a cistron. Thus, the cassettecomprises DNA expression control elements operatively linked to theupstream and downstream translatable DNA sequences. A cistron is formedwhen a translatable DNA sequence is directionally inserted(directionally ligated) between the upstream and downstream sequencesvia the sequence of nucleotides adapted for that purpose. The resultingthree translatable DNA sequences, namely the upstream, the inserted andthe downstream sequences, are all operatively linked in the same readingframe.

Thus, a DNA expression vector for expressing an antibody moleculeprovides a system for cloning translatable DNA sequences into thecassette portions of the vector to produce cistrons capable ofexpressing the first and second polypeptides, i.e., the heavy and lightchains of a monoclonal antibody.

As used herein, the term "vector" refers to a nucleic acid moleculecapable of transporting between different genetic environments anothernucleic acid to which it has been operatively linked. Preferred vectorsare those capable of autonomous replication and expression of structuralgene products present in the DNA segments to which they are operativelylinked. Vectors, therefore, preferably contain the replicons andselectable markers described earlier.

As used herein with regard to DNA sequences or segments, the phrase"operatively linked" means the sequences or segments have beencovalently joined, preferably by conventional phosphodiester bonds, intoone strand of DNA, whether in single or double stranded form. The choiceof vector to which transcription unit or a cassette of this invention isoperatively linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., vector replication and proteinexpression, and the host cell to be transformed, these being limitationsinherent in the art of constructing recombinant DNA molecules.

A sequence of nucleotides adapted for directional ligation, i.e., apolylinker, is a region of the DNA expression vector that (1)operatively links for replication and transport the upstream anddownstream translatable DNA sequences and (2) provides a site or meansfor directional ligation of a DNA sequence into the vector. Typically, adirectional polylinker is a sequence of nucleotides that defines two ormore restriction endonuclease recognition sequences, or restrictionsites. Upon restriction cleavage, the two sites yield cohesive terminito which a translatable DNA sequence can be ligated to the DNAexpression vector. Preferably, the two restriction sites provide, uponrestriction cleavage, cohesive termini that are non-complementary andthereby permit directional insertion of a translatable DNA sequence intothe cassette. In one embodiment, the directional ligation means isprovided by nucleotides present in the upstream translatable DNAsequence, downstream translatable DNA sequence, or both. In anotherembodiment, the sequence of nucleotides adapted for directional ligationcomprises a sequence of nucleotides that defines multiple directionalcloning means. Where the sequence of nucleotides adapted for directionalligation defines numerous restriction sites, it is referred to as amultiple cloning site.

In a preferred embodiment, a DNA expression vector is designed forconvenient manipulation in the form of a filamentous phage particleencapsulating a genome according to the teachings of the presentinvention. In this embodiment, a DNA expression vector further containsa nucleotide sequence that defines a filamentous phage origin ofreplication such that the vector, upon presentation of the appropriategenetic complementation, can replicate as a filamentous phage in singlestranded replicative form and be packaged into filamentous phageparticles. This feature provides the ability of the DNA expressionvector to be packaged into phage particles for subsequent segregation ofthe particle, and vector contained therein, away from other particlesthat comprise a population of phage particles.

A filamentous phage origin of replication is a region of the phagegenome, as is well known, that defines sites for initiation ofreplication, termination of replication and packaging of the replicativeform produced by replication (see, for example, Rasched et al.,Microbiol. Rev., 50: 401-427 (1986); and Horiuchi, J. Mol. Biol., 188:215-223 (1986)).

A preferred filamentous phage origin of replication for use in thepresent invention is an M13, f1 or fd phage origin of replication (Shortet al., Nucl. Acids Res., 16: 7583-7600 (1988)). Preferred DNAexpression vectors for cloning and expression a human monoclonalantibody of this invention are the dicistronic expression vectorspComb8, pComb2-8, pComb3, pComb2-3 and pComb2-3', described herein.

A particularly preferred vector of the present invention includes apolynucleotide sequence that encodes a heavy or light chain variableregion of a human monoclonal antibody of the present invention.Particularly preferred are vectors that include a nucleotide sequencethat encodes a heavy or light chain amino acid residue sequence shown inFIGS. 10-13, that encodes a heavy or light chain having the bindingspecificity of those sequences shown in FIGS. 10-13, or that encodes aheavy or light chain having conservative substitutions relative to asequence shown in FIGS. 10-13, and complementary polynucleotidesequences thereto.

Insofar as polynucleotides are component parts of a DNA expressionvector for producing a human monoclonal antibody heavy or light chainimmunoglobulin variable region amino acid residue sequence, theinvention also contemplates isolated polynucleotides that encode suchheavy or light chain sequences.

It is to be understood that, due to the genetic code and its attendantredundancies, numerous polynucleotide sequences can be designed thatencode a contemplated heavy or light chain immunoglobulin variableregion amino acid residue sequence. Thus, the invention contemplatessuch alternate polynucleotide sequences incorporating the features ofthe redundancy of the genetic code.

Insofar as the expression vector for producing a human monoclonalantibody of this invention is carried in a host cell compatible withexpression of the antibody, the invention contemplates a host cellcontaining a vector or polynucleotide of this invention. A preferredhost cell is E. coli, as described herein.

E. coli cultures containing preferred expression vectors that produce ahuman monoclonal antibody of this invention were deposited pursuant toBudapest Treaty requirements with the American Type Culture Collection(ATCC), Rockville, Md., as described herein.

EXAMPLES

The following examples are intended to illustrate, but not limit, thescope of the invention.

1. Construction of a Dicistronic Expression Vector for Producing aHeterodimeric Receptor on Phage Particles

To obtain a vector system for generating a large number of Fab antibodyfragments that can be screened directly, expression libraries inbacteriophage Lambda have previously been constructed as described inHuse et al., Science, 246: 1275-1281 (1989). These systems did notcontain design features that provide for the expressed Fab to betargeted to the surface of a filamentous phage particle.

The main criterion used in choosing a vector system was the necessity ofgenerating the largest number of Fab fragments which could be screeneddirectly. Bacteriophage Lambda was selected as the starting point todevelop an expression vector for three reasons. First, in vitropackaging of phage DNA was the most efficient method of reintroducingDNA into host cells. Second, it was possible to detect proteinexpression at the level of single phage plaques. Finally, the screeningof phage libraries typically involved less difficulty with nonspecificbinding. The alternative, plasmid cloning vectors, are only advantageousin the analysis of clones after they have been identified. Thisadvantage was not lost in the present system because of the use of adicistronic expression vector such as pCombVIII, thereby permitting aplasmid containing the heavy chain, light chain, or Fab expressinginserts to be excised.

a. Construction of Dicistronic Expression Vector pCOMB

1) Preparation of Lambda Zap ™II

Lambda Zap™ II is a derivative of the original Lambda Zap (ATCCAccession No. 40,298) that maintains all of the characteristics of theoriginal Lambda Zap including 6 unique cloning sites, fusion proteinexpression, and the ability to rapidly excise the insert in the form ofa phagemid (Bluescript SK-), but lacks the SAM 100 mutation, allowinggrowth on many Non-Sup F strains, including XL1-Blue. The Lambda Zap™ IIwas constructed as described in Short et al., Nuc. Acids Res., 16:7583-7600, 1988, by replacing the Lambda S gene contained in a 4254 basepair (bp) DNA fragment produced by digesting Lambda Zap with therestriction enzyme Nco I. This 4254 bp DNA fragment was replaced withthe 4254 bp DNA fragment containing the Lambda S gene isolated fromLambda gt10 (ATCC #40,179) after digesting the vector with therestriction enzyme Nco I. The 4254 bp DNA fragment isolated from lambdagt10 was ligated into the original Lambda Zap vector using T4 DNA ligaseand standard protocols such as those described in Current Protocols inMolecular Biology, Ausubel et al., eds., John Wiley and Sons, NY, 1987,to form Lambda Zap™ II.

2) Preparation of Lambda Hc2

To express a plurality of V_(H) -coding DNA homologs in an E. coli hostcell, a vector designated Lambda Hc2 was constructed. The vectorprovided the following: the capacity to place the V_(H) -coding DNAhomologs in the proper reading frame; a ribosome binding site asdescribed by Shine et al., Nature, 254: 34 (1975); a leader sequencedirecting the expressed protein to the periplasmic space designated thepelB secretion signal; a polynucleotide sequence that coded for a knownepitope (epitope tag); and also a polynucleotide that coded for a spacerprotein between the V_(H) -coding DNA homolog and the polynucleotidecoding for the epitope tag. Lambda Hc2 has been previously described byHuse et al., Science, 246: 1275-1281 (1989).

To prepare Lambda Hc2, a synthetic DNA sequence containing all of theabove features was constructed by designing single strandedpolynucleotide segments of 20-40 bases that would hybridize to eachother and form the double stranded synthetic DNA sequence shown inFIG. 1. The individual single-stranded polynucleotide segments are shownin Table 1.

Polynucleotides N2, N3, N9-4, Nil, N10-5, N6, N7 and N8 (Table 1) werekinased by adding 1 μl of each polynucleotide 0.1 micrograms/microliter(μg/μl) and 20 units of T₄ polynucleotide kinase to a solutioncontaining 70 mM Tris-HCl (Tris[hydroxymethyl] aminomethanehydrochloride) at pH 7.6, 10 mM MgCl₂, 5 mM dithiothreitol (DTT), 10 mMbeta-mercaptoethanol, 500 micrograms per milliliter (μg/ml) bovine serumalbumin (BSA). The solution was maintained at 37 degrees Centigrade (37°C.) for 30 minutes and the reaction stopped by maintaining the solutionat 65° C. for 10 minutes. The two end polynucleotides, 20 nanograms (ng)of polynucleotides N1 and polynucleotides N12, were added to the abovekinasing reaction solution together with 1/10 volume of a solutioncontaining 20 mM Tris-HCl at pH 7.4, 2.0 mM MgCl₂ and 50 mM NaCl. Thissolution was heated to 70° C. for 5 minutes and allowed to cool to roomtemperature, approximately 25° C., over 1.5 hours in a 500 ml beaker ofwater. During this time period all 10 polynucleotides annealed to formthe double stranded synthetic DNA insert shown in FIG. 1. The individualpolynucleotides were covalently linked to each other to stabilize thesynthetic DNA insert by adding 40 μl of the above reaction to a solutioncontaining 50 mM Tris-HCl at pH 7.5, 7 mM MgCl₂, 1 mM DTT, 1 mMadenosine triphosphate (ATP) and 10 units of T4 DNA ligase. Thissolution was maintained at 37° C. for 30 minutes and then the T4 DNAligase was inactivated by maintaining the solution at 65° C. for 10minutes. The end polynucleotides were kinased by mixing 52 μl of theabove reaction, 4 μl of a solution containing 10 mM ATP and 5 units ofT4 polynucleotide kinase. This solution was maintained at 37° C. for 30minutes and then the T4 polynucleotide kinase was inactivated bymaintaining the solution at 65° C. for 10 minutes.

                                      TABLE 1    __________________________________________________________________________    SEQ    ID NO    __________________________________________________________________________    (15)        N1) 5' GGCCGCAAATTCTATTTCAAGGAGACAGTCAT 3'    (16)        N2) 5' AATGAAATACCTATTGCCTACGGCAGCCGCTGGATT 3'    (17)        N3) 5' GTTATTACTCGCTGCCCAACCAGCCATGGCCC 3'    (18)        N6) 5' CAGTTTCACCTGGGCCATGGCTGGTTGGG 3'    (19)        N7) 5' CAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAG 3'    (20)        N8) 5' GTATTTCATTATGACTGTCTCCTTGAAATAGAATTTGC 3'    (21)        N9-4)            5' AGGTGAAACTGCTCGAGATTTCTAGACTAGTTACCCGTAC 3'    (22)        N10-5)            5' CGGAACGTCGTACGGGTAACTAGTCTAGAAATCTCGAG 3'    (23)        N11)            5' GACGTTCCGGACTACGGTTCTTAATAGAATTCG 3'    (24)        N12)            5' TCGACGAATTCTATTAAGAACCGTAGTC 3'    __________________________________________________________________________

The completed synthetic DNA insert was ligated directly into the LambdaZap™ II vector described in Example 1a1) that had been previouslydigested with the restriction enzymes, Not I and Xho I. The ligationmixture was packaged according to the manufacture's instructions usingGigapack II Gold packing extract available from Stratagene, La Jolla,Calif. The packaged ligation mixture was plated on XL1-Blue cells(Stratagene). Individual lambda plaques were cored and the insertsexcised according to the in vivo excision protocol for Lambda Zap™ IIprovided by the manufacturer (Stratagene). This in vivo excisionprotocol moved the cloned insert from the Lambda Hc2 vector into aphagemid vector to allow easy for manipulation and sequencing. Theaccuracy of the above cloning steps was confirmed by sequencing theinsert using the Sanger dideoxy method described in by Sanger et al.,Proc. Natl. Acad. Sci., USA, 74: 5463-5467 (1977) and using themanufacture's instructions in the AMV Reverse Transcriptase ³⁵ S-ATPsequencing kit (Stratagene). The sequence of the resultingdouble-stranded synthetic DNA insert in the V_(H) expression vector(Lambda Hc2) is shown in FIG. 1. The sequence of each strand (top andbottom) of Lambda Hc2 is listed in the Sequence Listing as SEQ ID NO 1and SEQ ID NO 2, respectively. The resultant Lambda Hc2 expressionvector is shown in FIG. 2.

3) Preparation of Lambda Lc2

To express a plurality of V_(L) -coding DNA homologs in an E. coli hostcell, a vector designated Lambda Lc2 was constructed having the capacityto place the V_(L) -coding DNA homologs in the proper reading frame,provided a ribosome binding site as described by Shine et al., Nature,254: 34 (1975), provided the pelB gene leader sequence secretion signalthat has been previously used to successfully secrete Fab fragments inE. coli by Lei et al., J. Bac., 169: 4379 (1987) and Better et al.,Science, 240: 1041 (1988), and also provided a polynucleotide containinga restriction endonuclease site for cloning. Lambda Lc2 has beenpreviously described by Huse et al., Science, 246: 1275-1281 (1989).

A synthetic DNA sequence containing all of the above features wasconstructed by designing single stranded polynucleotide segments of20-60 bases that would hybridize to each other and form the doublestranded synthetic DNA sequence shown in FIG. 3. The sequence of eachindividual single-stranded polynucleotide segment (01-08) within thedouble stranded synthetic DNA sequence is shown in Table 2.

Polynucleotides 02, 03, 04, 05, 06 and 07 (Table 2) were kinased byadding 1 μl (0.1 μg/μl) of each polynucleotide and 20 units of T₄polynucleotide kinase to a solution containing 70 mM Tris-HCl at pH 7.6,10 mM MgCl₂, 5 mM DTT, 10 mM beta-mercaptoethanol, 500 μg/ml of BSA. Thesolution was maintained at 37° C. for 30 minutes and the reactionstopped by maintaining the solution at 65° C. for 10 minutes. The 20 ngeach of the two end polynucleotides, 01 and 08, were added to the abovekinasing reaction solution together with 1/10 volume of a solutioncontaining 20.0 mM Tris-HCl at pH 7.4, 2.0 mM MgCl₂ and 15.0 mM sodiumchloride (NaCl). This solution was heated to 70° C. for 5 minutes andallowed to cool to room temperature, approximately 25° C., over 1.5hours in a 500 ml beaker of water. During this time period all 8polynucleotides annealed to form the double stranded synthetic DNAinsert shown in FIG. 3. The individual polynucleotides were covalentlylinked to each other to stabilize the synthetic DNA insert by adding 40μl of the above reaction to a solution containing 50 mM Tris-HCl at pH7.5, 7 mM MgCl₂, 1 mM DTT, 1 mM ATP and 10 units of T4 DNA ligase. Thissolution was maintained at 37° C. for 30 minutes and then the T4 DNAligase was inactivated by maintaining the solution at 65° C. for 10minutes. The end polynucleotides were kinased by mixing 52 μl of theabove reaction, 4 μl of a solution containing 10 mM ATP and 5 units ofT4 polynucleotide kinase. This solution was maintained at 37° C. for 30minutes and then the T4 polynucleotide kinase was inactivated bymaintaining the solution at 65° C. for 10 minutes.

                                      TABLE 2    __________________________________________________________________________    SEQ    ID NO    __________________________________________________________________________    (25)        01)          5' TGAATTCTAAACTAGTCGCCAAGGAGACAGTCAT 3'    (26)        02)          5' AATGAAATACCTATTGCCTACGGCAGCCGCTGGATT 3'    (27)        03)          5' GTTATTACTCGCTGCCCAACCAGCCATGGCC 3'    (28)        04)          5' GAGCTCGTCAGTTCTAGAGTTAAGCGGCCG 3'    (29)        05)          5' GTATTTCATTATGACTGTCTCCTTGGCGACTAGTTTAGAA-           TTCAAGCT 3'    (30)        06)          5' CAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAG 3'    (31)        07)          5' TGACGAGCTCGGCCATGGCTGGTTGGG 3'    (32)        08)          5' TCGACGGCCGCTTAACTCTAGAAC 3'    __________________________________________________________________________

The completed synthetic DNA insert was ligated directly into the LambdaZap™ II vector described in Example 1a1) that had been previouslydigested with the restriction enzymes Sac I and Xho I. The ligationmixture was packaged according to the manufacture's instructions usingGigapack II Gold packing extract (Stratagene). The packaged ligationmixture was plated on XL1-Blue cells (Stratagene). Individual lambdaplaques were cored and the inserts excised according to the in vivoexcision protocol for Lambda Zap™ II provided by the manufacturer(Stratagene). This in vivo excision protocol moved the cloned insertfrom the Lambda Lc2 vector into a plasmid phagemid vector allow for easymanipulation and sequencing. The accuracy of the above cloning steps wasconfirmed by sequencing the insert using the manufacture's instructionsin the AMV Reverse Transcriptase ³⁵ S-dATP sequencing kit (Stratagene).The sequence of the resulting Lc2 expression vector (Lambda Lc2) isshown in FIG. 3. Each strand is separately listed in the SequenceListing as SEQ ID NO 3 and SEQ ID NO 4. The resultant Lc2 vector isschematically diagrammed in FIG. 4.

A preferred vector for use in this invention, designated Lambda Lc3, isa derivative of Lambda Lc2 prepared above. Lambda Lc2 contains a Spe Irestriction site located 3' to the EcoR I restriction site and 5' to theShine-Dalgarno ribosome binding site as shown in the sequence in FIG. 3and in SEQ ID NO 3. A Spe I restriction site is also present in LambdaHc2 as shown in FIGS. 1 and 2 and in SEQ ID NO 1. A combinatorialvector, designated pComb, was constructed by combining portions ofLambda Hc2 and Lc2 together as described in Example 1a4) below. Theresultant combinatorial pComb vector contained two Spe I restrictionsites, one provided by Lambda Hc2 and one provided by Lambda Lc2, withan EcoR I site in between. Despite the presence of two Spe I restrictionsites, DNA homologs having Spe I and EcoR I cohesive termini weresuccessfully directionally ligated into a pComb expression vectorpreviously digested with Spe I and EcoR I as described in Example 1bbelow. The proximity of the EcoR I restriction site to the 3' Spe Isite, provided by the Lc2 vector, inhibited the complete digestion ofthe 3' Spe I site. Thus, digesting pComb with Spe I and EcoR I did notresult in removal of the EcoR I site between the two Spe I sites.

The presence of a second Spe I restriction site may be undesirable forligations into a pComb vector digested only with Spe I as the regionbetween the two sites would be eliminated. Therefore, a derivative ofLambda Lc2 lacking the second or 3' Spe I site, designated Lambda Lc3,was produced by first digesting Lambda Lc2 with Spe I to form alinearized vector. The ends were filled in to form blunt ends which areligated together to result in Lambda Lc3 lacking a Spe I site. LambdaLc3 is a preferred vector for use in constructing a combinatorial vectoras described below.

4) Preparation of pComb

Phagemids were excised from the expression vectors Lambda Hc2 or LambdaLc2 using an in vivo excision protocol described above. Double strandedDNA was prepared from the phagemid-containing cells according to themethods described by Holmes et al., Anal. Biochem., 114: 193 (1981). Thephagemids resulting from in vivo excision contained the same nucleotidesequences for antibody fragment cloning and expression as did the parentvectors, and are designated phagemid Hc2 and Lc2, corresponding toLambda Hc2 and Lc2, respectively.

For the construction of combinatorial phagemid vector pComb, produced bycombining portions of phagemid Hc2 and phagemid Lc2, phagemid Hc2 wasfirst digested with Sac I to remove the restriction site located 5' tothe LacZ promoter. The linearized phagemid was then blunt ended with T4polymerase and ligated to result in a Hc2 phagemid lacking a Sac I site.The modified Hc2 phagemid and the Lc2 phagemid were then separatelyrestriction digested with Sca I and EcoR I to result in a Hc2 fragmenthaving from 5' to 3' Sca I, Not I, Xho I, Spe I and EcoR I restrictionsites and a Lc2 fragment having from 5' to 3' EcoR I, Sac I, Xba I andSac I restriction sites. The linearized phagemids were then ligatedtogether at their respective cohesive ends to form pComb, a circularizedphagemid having a linear arrangement of restriction sites of Not I, XhoI, Spe I, EcoR I, Sac I, Xba I, Not I, Apa I and Sca I. The ligatedphagemid vector was then inserted into an appropriate bacterial host andtransformants were selected on the antibiotic ampicillin.

Selected ampicillin resistant transformants were screened for thepresence of two Not I sites. The resulting ampicillin resistantcombinatorial phagemid vector was designated pComb, the schematicorganization of which is shown in FIG. 5. The resultant combinatorialvector, pComb, consisted of a DNA molecule having two cassettes toexpress two fusion proteins and having nucleotide residue sequences forthe following operatively linked elements listed in a 5' to 3'direction: a first cassette consisting of an inducible LacZ promoterupstream from the LacZ gene; a Not I restriction site; a ribosomebinding site; a pelB leader; a spacer; a cloning region bordered by a 5'Xho and 3' Spe I restriction site; a decapeptide tag followed byexpression control stop sequences; an EcoR I restriction site located 5'to a second cassette consisting of an expression control ribosomebinding site; a pelB leader; a spacer region; a cloning region borderedby a 5' Sac I and a 3' Xba I restriction site followed by expressioncontrol stop sequences and a second Not I restriction site.

A preferred combinatorial vector for use in this invention, designatedpComb2, is constructed by combining portions of phagemid Hc2 andphagemid Lc3 as described above for preparing pComb. The resultantcombinatorial vector, pComb2, consists of a DNA molecule having twocassettes identical to pComb to express two fusion proteins identicallyto pComb except that a second Spe I restriction site in the secondcassette is eliminated.

b. Construction of the pCombIII Vector for Expressing Fusion ProteinsHaving a Bacteriophage Coat Protein Membrane Anchor

Because of the multiple endonuclease restriction cloning sites, thepComb phagemid expression vector prepared above is a useful cloningvehicle for modification for the preparation of an expression vector foruse in this invention. To that end, pComb was digested with EcoR I andSpe I followed by phosphatase treatment to produce linearized pComb.

1) Preparation of pCombIII

A separate phagemid expression vector was constructed using sequencesencoding bacteriophage cpIII membrane anchor domain. A PCR productdefining the DNA sequence encoding the filamentous phage coat protein,cpIII, membrane anchor containing a LacZ promotor region sequence 3' tothe membrane anchor for expression of the light chain and Spe I and EcoRI cohesive termini was prepared from M13mp18, a commercially availablebacteriophage vector (Pharmacia, Piscataway, N.J.).

To prepare a modified cpIII, replicative form DNA from M13mp18 was firstisolated. Briefly, into 2 ml of LB (Luria-Bertani medium), 50 μl of aculture of a bacterial strain carrying an F' episome (JM107, JM109 orTG1) was admixed with a one tenth suspension of bacteriophage particlesderived from a single plaque. The admixture was incubated for 4 to 5hours at 37° C. with constant agitation. The admixture was thencentrifuged at 12,000×g for 5 minutes to pellet the infected bacteria.After the supernatant was removed, the pellet was resuspended byvigorous vortexing in 100 μl of ice-cold solution I. Solution I Wasprepared by admixing 50 mM glucose, 10 mM EDTA (disodiumethylenediaminetetraacetic acid) and 25 mM Tris-HCl at pH 8.0, andautoclaving for 15 minutes.

To the bacterial suspension, 200 μl of freshly prepared Solution II wasadmixed and the tube was rapidly inverted five times. Solution II wasprepared by admixing 0.2N NaOH and 1% SDS. To the bacterial suspension,150 μl of ice-cold Solution III was admixed and the tube was vortexedgently in an inverted position for 10 seconds to disperse Solution IIIthrough the viscous bacterial lysate. Solution III was prepared byadmixing 60 ml of 5M potassium acetate, 11.5 ml of glacial acetic acidand 28.5 ml of water. The resultant bacterial lysate was then stored onice for 5 minutes followed by centrifugation at 12,000×g for 5 minutesat 4° C. in a microfuge. The resultant supernatant was recovered andtransferred to a new tube. To the supernatant was added an equal volumeof phenol/chloroform and the admixture was vortexed. The admixture wasthen centrifuged at 12,000×g for 2 minutes in a microfuge. The resultantsupernatant was transferred to a new tube and the double-strandedbacteriophage DNA was precipitated with 2 volumes of ethanol at roomtemperature. After allowing the admixture to stand at room temperaturefor 2 minutes, the admixture was centrifuged to pellet the DNA. Thesupernatant was removed and the pelleted replicative form DNA wasresuspended in 25 μl of Tris-HCl at pH 7.6, and 10 mM EDTA (TE).

An alternative Lac-B primer for use in constructing the cpIII membraneanchor and LacZ promotor region was Lac-B' as shown in Table 3. Theamplification reactions were performed as described above with theexception that in the second PCR amplification, Lac-B' was used withLac-F instead of Lac-B. The product from the amplification reaction islisted in the sequence listing as SEQ ID NO 41 from nucleotide position1 to nucleotide position 172. The use of Lac-B' resulted in a LacZregion lacking 29 nucleotides on the 3' end but was functionallyequivalent to the longer fragment produced with the Lac-F and Lac-Bprimers.

The products of the first and second PCR amplifications using the primerpairs G-3(F) and G-3(B) and Lac-F and Lac-B were then recombined at thenucleotides corresponding to cpIII membrane anchor overlap and Nhe Irestriction site and subjected to a second round of PCR using the G-3(F)(SEQ ID NO 35) and Lac-B (SEQ ID NO 38) primer pair to form a recombinedPCR DNA fragment product consisting of the following: a 5' Spe Irestriction site; a cpIII DNA membrane anchor domain beginning at thenucleotide residue sequence which corresponds to the amino acid residue198 of the entire mature cpIII protein; an endogenous stop site providedby the membrane anchor at amino acid residue number 112; a Nhe Irestriction site, a LacZ promoter, operator and Cap-binding sitesequence; and a 3' EcoR I restriction site.

To construct a phagemid vector for the coordinate expression of a heavychain-cpIII fusion protein as prepared in Example 2 with kappa lightchain, the recombined PCR modified cpIII membrane anchor domain DNAfragment was then restriction digested with Spe I and EcoR I to producea DNA fragment for directional ligation into a similarly digested pComb2phagemid expression vector having only one Spe I site prepared inExample 1a4) to form a pComb2-III (also referred to as pComb2-III)phagemid expression vector. Thus, the resultant ampicillin resistanceconferring pComb2-3 vector, having only one Spe I restriction site,contained separate LacZ promoter/operator sequences for directing theseparate expression of the heavy chain (Fd)-cpIII fusion product and thelight chain protein. The expressed proteins were directed to theperiplasmic space by pelB leader sequences for functional assembly onthe membrane. Inclusion of the phage F1 intergenic region in the vectorallowed for packaging of single stranded phagemid with the aid of helperphage. The use of helper phage superinfection lead to expression of twoforms of cpIII. Thus, normal phage morphogenesis was perturbed bycompetition between the Fab-cpIII fusion and the native cpIII of thehelper phage for incorporation into the virion for Fab-cpVIII fusions.In addition, also contemplated for use in this invention are vectorsconferring chloramphenicol resistance and the like.

A more preferred phagemid expression vector for use in this inventionhaving additional restriction enzyme cloning sites, designatedpComb-III' or pComb2-3', was prepared as described above for pComb2-3with the addition of a 51 base pair fragment from pBluescript asdescribed by Short et al., Nuc. Acids Res., 16: 7583-7600 (1988) andcommercially available from Stratagene. To prepare pComb2-3', pComb2-3was first digested with Xho I and Spe I restriction enzymes to form alinearized pComb2-3. The vector pBluescript was digested with the sameenzymes releasing a 51 base pair fragment containing the restrictionenzyme sites Sal I, Acc I, Hinc II, Cla I, Hind III, EcoR V, Pst I, SmaI and BamH I. The 51 base pair fragment was ligated into the linearizedpComb2-3 vector via the cohesive Xho I and Spe I termini to formpComb2-3'.

                                      TABLE 3    __________________________________________________________________________    SEQ ID NO          Primer    __________________________________________________________________________    (35).sup.1          G-3 (F)               5' GAGACGACTAGTGGTGGCGGTGGCTCTCCATTC                GTTTGTGAATATCAA 3'    (36).sup.2          G-3 (B)               5' TTACTAGCTAGCATAATAACGGAATACCCAAAA                GAACTGG 3'    (37).sup.3          LAC-F               5' TATGCTAGCTAGTAACACGACAGGTTTCCCGAC                TGG 3'    (38).sup.4          LAC-B               5' ACCGAGCTCGAATTCGTAATCATGGTC 3'    (39)5 LAC-B'               5' AGCTGTTGAATTCGTGAAATTGTTATCCGCT 3'    __________________________________________________________________________     F Forward Primer     B Backward Primer     1 From 5' to 3': Spe I restriction site sequence is single underlined; th     overlapping sequence with the 5' end of cpIII is double underlined     2 From 5' to 3': Nhe I restriction site sequence is single underlined; th     overlapping sequence with 3' end of cpIII is double underlined.     3 From 5' to 3': overlapping sequence with the 3' end of cpIII is double     underlined; Nhe I restriction sequence begins with the nucleotide residue     "G" at position 4 and extends 5 more residues = GCTAGC.     4 EcoR I restriction site sequence is single underlined.     5 Alternative backwards primer for amplifying LacZ; EcoR I restriction     site sequence is single underlined.

2. Isolation of HIV-1-Specific Monoclonal Antibodies Produced from theDicistronic Expression Vector, pComb2-3

In practicing this invention, the heavy (Fd consisting of V_(H) andC_(H) 1) and light (kappa) chains (V_(L), C_(L)) of antibodies are firsttargeted to the periplasm of E. coli for the assembly of heterodimericFab molecules. In order to obtain expression of antibody Fab librarieson a phage surface, the nucleotide residue sequences encoding either theFd or light chains must be operatively linked to the nucleotide residuesequence encoding a filamentous bacteriophage coat protein membraneanchor. A coat protein for use in this invention in providing a membraneanchor is III (cpIII or cp3). In the Examples described herein, methodsfor operatively linking a nucleotide residue sequence encoding a Fdchain to a cpIII membrane anchor in a fusion protein of this inventionare described.

In a phagemid vector, a first and second cistron consisting oftranslatable DNA sequences are operatively linked to form a dicistronicDNA molecule. Each cistron in the dicistronic DNA molecule is linked toDNA expression control sequences for the coordinate expression of afusion protein, Fd-cpIII, and a kappa light chain.

The first cistron encodes a periplasmic secretion signal (pelB leader)operatively linked to the fusion protein, Fd-cpIII. The second cistronencodes a second pelB leader operatively linked to a kappa light chain.The presence of the pelB leader facilitates the coordinated but separatesecretion of both the fusion protein and light chain from the bacterialcytoplasm into the periplasmic space.

In this process, the phagemid expression vector carries an ampicillinselectable resistance marker gene (beta lactamase or bla) in addition tothe Fd-cpIII fusion and the kappa chain. The f1 phage origin ofreplication facilitates the generation of single stranded phagemid. Theisopropyl thiogalactopyranoside (IPTG) induced expression of adicistronic message encoding the Fd-cpIII fusion (V_(H), C_(H1), cpIII)and the light chain (V_(L), C_(L)) leads to the formation of heavy andlight chains. Each chain is delivered to the periplasmic space by thepelB leader sequence, which is subsequently cleaved. The heavy chain isanchored in the membrane by the cpIII membrane anchor domain while thelight chain is secreted into the periplasm. The heavy chain in thepresence of light chain assembles to form Fab molecules. This sameresult can be achieved if, in the alternative, the light chain isanchored in the membrane via a light chain fusion protein having amembrane anchor and heavy chain is secreted via a pelB leader into theperiplasm.

With subsequent infection of E. coli with a helper phage, as theassembly of the filamentous bacteriophage progresses, the coat proteinIII is incorporated on the tail of the bacteriophage.

a. Preparation of Lymphocyte RNA

Five milliliters of bone marrow was removed by aspiration from HIV-1asymptomatic seropositive individuals. Total cellular RNA was preparedfrom the bone marrow lymphocytes as described above using the RNApreparation methods described by Chomczynski et al., Anal Biochem., 162:156-159 (1987) and using the RNA isolation kit (Stratagene) according tothe manufacturer's instructions. Briefly, for immediate homogenizationof the cells in the isolated bone marrow, 10 ml of a denaturing solutioncontaining 3.0M guanidinium isothiocyanate containing 71 μl ofbeta-mercaptoethanol was admixed to the isolated bone marrow. One ml ofsodium acetate at a concentration of 2M at pH 4.0 was then admixed withthe homogenized cells. One ml of phenol that had been previouslysaturated with H₂ O was also admixed to the denaturing solutioncontaining the homogenized spleen. Two ml of a chloroform:isoamylalcohol (24:1 v/v) mixture was added to this homogenate. The homogenatewas mixed vigorously for ten seconds and maintained on ice for 15minutes. The homogenate was then transferred to a thick-walled 50 mlpolypropylene centrifuged tube (Fisher Scientific Company, Pittsburgh,Pa.). The solution was centrifuged at 10,000×g for 20 minutes at 4° C.The upper RNA-containing aqueous layer was transferred to a fresh 50 mlpolypropylene centrifuge tube and mixed with an equal volume ofisopropyl alcohol. This solution was maintained at -20° C. for at leastone hour to precipitate the RNA. The solution containing theprecipitated RNA was centrifuged at 10,000×g for twenty minutes at 4° C.The pelleted total cellular RNA was collected and dissolved in 3 ml ofthe denaturing solution described above. Three ml of isopropyl alcoholwas added to the re-suspended total cellular RNA and vigorously mixed.This solution was maintained at -20° C. for at least 1 hour toprecipitate the RNA. The solution containing the precipitated RNA wascentrifuged at 10,000×g for ten minutes at 4° C. The pelleted RNA waswashed once with a solution containing 75% ethanol. The pelleted RNA wasdried under vacuum for 15 minutes and then re-suspended in dimethylpyrocarbonate-treated (DEPC-H₂ O) H₂ O.

Messenger RNA (mRNA) enriched for sequences containing long poly Atracts was prepared from the total cellular RNA using methods describedin Molecular Cloning: A Laboratory Manual, Maniatis et al., eds., ColdSpring Harbor, N.Y., (1982). Briefly, one half of the total RNA isolatedfrom a single donor prepared as described above was resuspended in oneml of DEPC-H₂ O and maintained at 65° C. for five minutes. One ml of 2×high salt loading buffer consisting of 100 mM Tris-HCl, 1M NaCl, 2.0 mMEDTA at pH 7.5, and 0.2% SDS was admixed to the resuspended RNA and themixture allowed to cool to room temperature.

The total purified mRNA was then used in PCR amplification reactions asdescribed in Example 2c. Alternatively, the mRNA was further purified topoly A+ RNA by the following procedure. The total MRNA was applied to anoligo-dT (Collaborative Research Type 2 or Type 3) column that waspreviously prepared by washing the oligo-dT with a solution containing0.1M sodium hydroxide and 5 mM EDTA and then equilibrating the columnwith DEPC-H₂ O. The eluate was collected in a sterile polypropylene tubeand reapplied to the same column after heating the eluate for 5 minutesat 65° C. The oligo-dT column was then washed with 2 ml of high saltloading buffer consisting of 50 mM Tris-HCl at pH 7.5, 500 mM sodiumchloride, 1 mM EDTA at pH 7.5 and 0.1% SDS. The oligo dT column was thenwashed with 2 ml of 1× medium salt buffer consisting of 50 mM Tris-HCl,pH 7.5, 100 mM, 1 mM EDTA and 0.1% SDS. The messenger RNA was elutedfrom the oligo-dT column with 1 ml of buffer consisting of 10 mMTris-HCl at pH 7.5, 1 mM EDTA at pH 7.5, and 0.05% SDS. The messengerRNA was purified by extracting this solution with phenol/chloroformfollowed by a single extraction with 100% chloroform. The messenger RNAwas concentrated by ethanol precipitation and resuspended in DEPC

The resultant purified mRNA contained a plurality of anti-HIV encodingV_(H) and V_(L) sequences for preparation of an anti-HIV-1 Fab DNAlibrary.

b. Construction of a Combinatorial HIV-1 Antibody Library

1) Selection of Oligonucleotide Primers

The nucleotide sequences encoding the immunoglobulin protein CDR's arehighly variable. However, there are several regions of conservedsequences that flank the V region domains of either the light or heavychain, for instance, and that contain substantially conserved nucleotidesequences, i.e., sequences that will hybridize to the same primersequence. Therefore, polynucleotide synthesis (amplification) primersthat hybridize to the conserved sequences and incorporate restrictionsites into the DNA homolog produced that are suitable for operativelylinking the synthesized DNA fragments to a vector were constructed. Morespecifically, the primers were designed so that the resulting DNAhomologs produced can be inserted into an expression vector of thisinvention in reading frame with the upstream translatable DNA sequenceat the region of the vector containing the directional ligation means.

For amplification of the V_(H) domains, primers were designed tointroduce cohesive termini compatible with directional ligation into theunique Xho I and Spe I sites of the pComb2-3 expression vector. In allcases, the 5' primers VH1a (5' CAGGTGCAGCTCGAGCAGTCTGGG 3' SEQ ID NO 42)and VH3a (5' GAGGTGCAGCTCGAGGAGTCTGGG 3' SEQ ID NO 43) were designed tomaximize homology with the V_(H) 1 and V_(H) 3 subgroup families,respectively, although considerable cross-priming of other subgroups wasexpected. The Xho I restriction site for cloning into the pComb2-3vector is underlined. The 3' primer CG1z having the nucleotide sequence5' GCATGTACTAGTTTTGTCACAAGATTTGGG 3' (SEQ ID NO 44) used in conjunctionwith the 5' primers is the primer for the heavy chain corresponding topart of the hinge region. The Spe I site for cloning into the pComb2-3vector is underlined.

The nucleotide sequences encoding the V_(L) domain are highly variable.However, there are several regions of conserved sequences that flank theV_(L) domains including the J_(L), V_(L) framework regions and V_(L)leader/promotor. Therefore, amplification primers were constructed thathybridized to the conserved sequences and incorporate restriction sitesthat allow cloning the amplified fragments into the pComb2-3 expressionvector cut with Sac I and Xba I.

For amplification of the kappa V_(L) domains analogous to the heavychain primers listed above, the 5' primers, VK1a (5'GACATCGAGCTCACCCAGTCTCCA 3' SEQ ID NO 45) and VK3a (5'GAAATTGAGCTCACGCAGTCTCCA 3' SEQ ID NO 46), were used. These primers alsointroduced a Sac I restriction endonuclease site indicated by theunderlined nucleotides to allow the V_(L) DNA homolog to be cloned intothe pComb2-3 expression vector. The 3' V_(L) amplification primer, CK1ahaving a nucleotide sequence 5'GCGCCGTCTAGAACTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCAAG 3' (SEQ ID NO 47)corresponding to the 3' end of the light chain was used to amplify thelight chain while incorporating the underlined Xba I restrictionendonuclease site required to insert the V_(L) DNA homolog into thepComb2-3 expression vector.

All primers and synthetic polynucleotides described herein, were eitherpurchased from Research Genetics in Huntsville, Alabama or synthesizedon an Applied Biosystems DNA synthesizer, model 381A, using themanufacturer's instruction.

2) PCR Amplification of V_(H) and V_(L) DNA Homologs

In preparation for PCR amplification, mRNA prepared above was used as atemplate for cDNA synthesis by a primer extension reaction. First, 20-50μg of total mRNA in water was first hybridized (annealed) at 70° C. for10 minutes with 600 ng (60.0 pmol) of either the heavy or light chain 3'primers listed above. Subsequently, the hybridized admixture was used ina typical 50 μl reverse transcription reaction containing 200 μM each ofdATP, dCTP, dGTP and dTTP, 40 mM Tris-HCl at pH 8.0, 8 mM MgCl₂, 50 mMNaCl, 2 mM spermidine and 600 units of reverse transcriptase(SuperScript, BRL). The reaction admixture was then maintained for onehour at 37° C. to form an RNA-cDNA admixture.

Three μl of the resultant RNA-cDNA admixture was then used in PCRamplification in a reaction volume of 100 μl containing a mixture of allfour dNTPs at a concentration of 60 μM, 50 mM KCl, 10 mM Tris-HCl at pH8.3, 15 mM MgCl₂, 0.1% gelatin and 5 units of Thermus aquaticus (Taq)DNA polymerase (Perkin-Elmer-Cetus, Emeryville, Calif.), and 60 pmol ofthe appropriate 5' and 3' primers listed above. The separate reactionadmixtures were overlaid with mineral oil and subjected to 35 cycles ofamplification. Each amplification cycle included denaturation at 91° C.for 1 minute, annealing at 52° C. for 2 minutes and polynucleotidesynthesis by primer extension (elongation) at 72° C. for 1.5 minutes,followed by a final maintenance period of 10 minutes at 72° C. Analiquot of the reaction admixtures were then separately electrophoresedon a 2% agarose gel. After successful amplification as determined by gelelectrophoretic migration, the remainder of the RNA-cDNA was amplifiedafter which the PCR products of a common 3' primer were pooled intoseparate V_(H) -and V_(L) -coding DNA homolog-containing samples andwere then extracted twice with phenol/chloroform, once with chloroform,ethanol precipitated and were stored at -70° C. in 10 mM Tris-HCl at pH7.5, and 1 mM EDTA.

3) Insertion of V_(H) and V_(L) -Coding DNA Homologs into pComb2-3Expression Vector

The V_(H) -coding DNA homologs (heavy chain) prepared above were thendigested with an excess of Xho I and Spe I for subsequent ligation intoa similarly digested and linearized pComb2-3 in a total volume of 150 μlwith 10 units of ligase at 16° C. overnight. The construction of thelibrary was performed as described by Burton et al., Proc. Natl. Acad.Sci., USA, 88: 10134-10137 (1991). Briefly, following ligation, thepComb2-3 vector containing heavy chain DNA was then transformed byelectroporation into 300 μl of XL1-Blue cells. After transformation andculturing, library size was determined by plating aliquots of theculture. Typically the library had about 10⁷ members. An overnightculture was then prepared from which phagemid DNA containing the heavychain library was prepared.

For the cloning of the V_(L) -coding DNA homologs (light chain), 10 μgof phagemid DNA containing the heavy chain library was then digestedwith Sac I and SbaI. The resulting linearized vector was treated withphosphatase and purified by agarose gel electrophoresis. The desiredfragment, 4.7 kb in length, was excised from the gel. Ligation of thisvector with prepared light chain PCR DNA proceeded as described abovefor heavy chain. A library of approximately 10⁷ members having heavychain fragments operatively linked to the cpIII anchor sequence(Fd-cpIII) and light chain fragments was thus produced.

4) Preparation of Phage Expressing Fab Heterodimers

Following transformation of the resultant library produced above intoXL1-Blue cells, phage were prepared to allow for isolation of HIV-1specific Fabs by panning on target antigens. To isolate phage on whichheterodimer expression has been induced, 3 ml of SOC medium (SOC wasprepared by admixture of 20 g bacto-tryptone, 5 g yeast extract and 0.5g NaCl in one liter of water, adjusting the pH to 7.5 and admixing 20 mlof glucose just before use to induce the expression of the Fd-cpIII andlight chain heterodimer) was admixed and the culture was shaken at 220rpm for one hour at 37° C., after which time 10 ml of SB (SB wasprepared by admixing 30 g tryptone, 20 g yeast extract, and 10 g Mopsbuffer per liter with pH adjusted to 7) containing 20 μg/mlcarbenicillin and 10 μg/ml tetracycline and the admixture was shaken at300 rpm for an additional hour. This resultant admixture was admixed to100 ml SB containing 50 μg/ml carbenicillin and 10 μg/ml tetracyclineand shaken for one hour, after which time helper phage VCSM13 (10¹² pfu)were admixed and the admixture was shaken for an additional two hours.After this time, 70 μg/ml kanamycin was admixed and maintained at 30° C.overnight. The lower temperature resulted in better heterodimerincorporation on the surface of the phage. The supernatant was clearedby centrifugation (4000 rpm for 15 minutes in a JA10 rotor at 4° C.).Phage were precipitated by admixture of 4% (w/v) polyethylene glycol8000 and 3% (w/v) NaCl and maintained on ice for 30 minutes, followed bycentrifugation (9000 rpm for 20 minutes in a JA10 rotor at 4° C.). Phagepellets were resuspended in 2 ml of PBS and microcentrifuged for threeminutes to pellet debris, transferred to fresh tubes and stored at -20°C. for subsequent screening as described below.

For determining the titering colony forming units (cfu), phage (packagedphagemid) were diluted in SB and 1 μl was used to infect 50 μl of fresh(OD600=1) XL1-Blue cells grown in SB containing 10 μg/ml tetracycline.Phage and cells were maintained at room temperature for 15 minutes andthen directly plated on LB/carbenicillin plates.

5) Selection of Anti-HIV-1 Heterodimers on Phage Surfaces

(a) Multiple Pannings of the Phage Library

The phage library produced in Example 2b4) was panned againstrecombinant gp120 of HIV-1 strain IIIb as described herein on coatedmicrotiter plate to select for anti-gp120 heterodimers. A second phagelibrary was panned against recombinant gp41 (American Biotechnologies,Boston, Mass.) as described below to select for anti-gp41 heterodimers.

The panning procedure used was a modification of that originallydescribed by Parmley and Smith (Parmley et al., Gene, 73: 305-318(1988). Four rounds of panning were performed to enrich for specificantigen-binding clones. For this procedure, four wells of a microtiterplate (Costar 3690) were coated overnight at 4° C. with 25 μl of 40μg/ml gp120 or gp41 (American Biotechnologies) prepared above in 0.1Mbicarbonate, pH 8.6. The wells were washed twice with water and blockedby completely filling the well with 3% (w/v) BSA in PBS and maintainingthe plate at 37° C. for one hour. After the blocking solution was shakenout, 50 μl of the phage library prepared above (typically 10¹¹ cfu) wereadmixed to each well, and the plate was maintained for two hours at 37°C.

Phage were removed and the plate was washed once with water. Each wellwas then washed ten times with TBS/Tween (50 mM Tris-HCl at pH 7.5, 150mM NaCl, 0.5% Tween 20) over a period of one hour at room temperaturewhere the washing consisted of pipetting up and down to wash the well,each time allowing the well to remain completely filled with TBS/Tweenbetween washings. The plate was washed once more with distilled waterand adherent phage were eluted by the addition of 50 μl of elutionbuffer (0.1M HCl, adjusted to pH 2.2 with solid glycine, containing 1mg/ml BSA) to each well followed by maintenance at room temperature for10 minutes. The elution buffer was pipetted up and down several times,removed, and neutralized with 3 μl of 2M Tris base per 50 μl of elutionbuffer used.

Eluted phage were used to infect 2 ml of fresh (OD₆₀₀ =1) E. coliXL1-Blue cells for 15 minutes at room temperature, after which time 10ml of SB containing 20 μg/ml carbenicillin and 10 μg/ml tetracycline wasadmixed. Aliquots of 20, 10, and 1/10 μl were removed from the culturefor plating to determine the number of phage (packaged phagemids) thatwere eluted from the plate. The culture was shaken for one hour at 37°C., after which it was added to 100 ml of SB containing 50 μg/mlcarbenicillin and 10 μg/ml tetracycline and shaken for one hour. Helperphage VCSM13 (10¹² pfu) were then added and the culture was shaken foran additional two hours. After this time, 70 μg/ml kanamycin was addedand the culture was incubated at 37° C. overnight. Phage preparation andfurther panning were repeated as described above.

Following each round of panning, the percentage yield of phage weredetermined, where % yield--(number of phage eluted/number of phageapplied) X 100. The initial phage input ratio was determined by titeringon selective plates to be approximately 10¹¹ cfu for each round ofpanning. The final phage output ratio was determined by infecting two mlof logarithmic phase XL1-Blue cells as described above and platingaliquots on selective plates. In the first panning for gp120-reactivephage, 4.6×10¹¹ phage were applied to four wells and 7.7×10⁵ phage wereeluted. After the fourth panning 1.0×10⁸ phage were eluted. From thisprocedure, 20 clones were selected from the Fab library for theirability to bind to glycosylated recombinant gp120 from the IIIB strainof HIV-1. Five clones were selected from the Fab library specific forbinding to gp41. The panned phage surface libraries were then convertedinto ones expressing soluble Fab fragments for further screening byELISA as described below.

In addition to panning on gp120 of strain IIIB and gp41, alsocontemplated as antigens for panning of combinatorial libraries isrecombinant gp120 (IIIB strain) produced in baculovirus and recombinantgp120 (SF2 strain) produced in Chinese Hamster Ovary cells obtained asdescribed by Steimer et al., Science, 254: 105-108 (1991). Anotherantigen, a synthetic cyclic peptide, N═CH--(CH₂)₃ CO[SISGPGRAFYTG]NCH₂CO--Cys --NH₂ (SEQ ID NO 48) prepared as described by Satterthwait etal., Bulletin of the World Health Organization, 68: Suppl., 17-25 (1990)corresponding to the central most conserved part of the V3 loop of gp120was coupled to maleimide-activated BSA. The library was panned using 1,2 or 4 ELISA wells coated with 1 μg of protein antigen or 10 μgBSA-peptide per well. Four rounds of panning were carried out for eachantigen as described above. Eluted phage from the final round were usedto infect XL1-Blue cells. Four rounds of panning against the fourantigens produced an amplification in eluted phage of between 100 and1000 fold. The panned phage surface libraries were then converted intoones expressing soluble Fab fragments for further screening by ELISA asdescribed below.

6) Preparation of Soluble Heterodimers and Characterization of BindingSpecificity to HIV-1 Antigens

In order to further characterize the specificity of the mutagenizedheterodimers expressed on the surface of phage as described above,soluble Fab heterodimers from acid eluted phage were prepared andanalyzed in ELISA assays on HIV-1 derived antigen-coated plates and bycompetitive ELISA.

To prepare soluble heterodimers, phagemid DNA from the 20 gp120 positiveclones and the 5 gp41 positive clones prepared above was isolated anddigested with Spe I and Nhe I. Digestion with these enzymes producedcompatible cohesive ends. The 4.7 kb DNA fragment lacking the gene IIIportion was gel-purified (0.6% agarose) and self-ligated. Transformationof E. coli XL1-Blue afforded the isolation of recombinants lacking thecpIII fragment. Clones were examined for removal of the cpIII fragmentby Xho I-Xba I digestion, which should yield an 1.6-kb fragment. Cloneswere grown in 100 ml SB containing 50 μg/ml carbenicillin and 20 mMMgCl₂ at 37° C. until an OD₆₀₀ of 0.2 was achieved. IPTG (1 mM) wasadded and the culture grown overnight at 30° C. (growth at 37° C.provides only a light reduction in heterodimer yield). Cells werepelleted by centrifugation at 4000 rpm for 15 minutes in a JA10 rotor at4° C. Cells were resuspended in 4 ml PBS containing 34 μ/mlphenylmethylsulfonyl fluoride (PMSF) and lysed by sonication on ice (2-4minutes at 50% duty). Debris was pelleted by centrifugation at 14,000rpm in a JA20 rotor at 4° C. for 15 minutes. The supernatant was useddirectly for ELISA analysis as described below and was stored at -20° C.For the study of a large number of clones, 10 ml cultures providedsufficient heterodimer for analysis. In this case, sonications wereperformed in 2 ml of buffer. Assays as described above were alsoperformed for the gp41-specific clones.

a) Screening by ELISA

The soluble heterodimers prepared above were assayed by ELISA. For thisassay, gp120 and gp41 were separately admixed to individual wells of amicrotiter plate as described above for the panning procedure andmaintained at 4° C. overnight to allow the protein solution to adhere tothe walls of the well. After the maintenance period, the wells werewashed five times with water and thereafter maintained for one hour at37° C. with 100 μl solution of 1% BSA diluted in PBS to blocknonspecific sites on the wells. Afterwards, the plates were inverted andshaken to remove the BSA solution. Twenty-five μl of solubleheterodimers prepared above reactive with the specific glycoproteinsubstrate were then admixed to each well and maintained at 37° C. forone hour to form immunoreaction products. Following the maintenanceperiod, the wells were washed ten times with water to remove unboundsoluble antibody and then maintained with a 25 μl of a 1:1000 dilutionof secondary goat anti-human IgG F(ab')₂ conjugated to alkalinephosphatase diluted in PBS containing 1% BSA. The wells were maintainedat 37° C. for one hour after which the wells were washed ten times withwater followed by development with 50 μl of p-nitrophenyl phosphate(PNPP). Color development was monitored at 405 nm. Positive clones gaveA405 values of >1 (mostly >1.5) after 10 minutes, whereas negativeclones gave values of 0.1to0.2.

Approximate concentrations of gp120-reactive Fab were determined byELISA using a sandwich ELISA as described by Zebedee et al., Proc. Natl.Acad. Sci., USA, 89: 3175-3179 (1992) and are presented in the firstcolumn of FIG. 6. In addition, since Fabs are expressed in E. coli andthe fraction of correctly assemble protein can vary, the amount of Fabreacting with gp120 was also assessed by ELISA titration. That data isalso presented in FIG. 6 in the second column.

For the clones panned against the HIV-1 derived antigens, afterconversion of the panned phage surface libraries to ones expressingsoluble Fab fragments, 30-40 colonies were used to transform XL1-Bluecells and the supernates screened in ELISA assays against the antigenused in panning. Generally greater than 80% of the supernates testedpositive. A representative number of positives were then selected fromeach antigen panning for further analysis.

(b) Competitive ELISA with Soluble gp120 and CD4

Immunoreactive heterodimers as determined in the above ELISA were thenanalyzed by competition ELISA to determine the affinity of the selectedheterodimers. The ELISA was performed as described above on microtiterwells separately coated with 5 μg/ml of gp120 or soluble CD4 (AmericanBiotechnologies) in 0.1M bicarbonate buffer at pH 8.6. Increasingconcentrations of soluble or free gp120 ranging in concentration from10⁻¹¹ M up to 10⁻⁷ M diluted in 0.5% BSA/0.025% Tween 20/PBS wereadmixed with soluble heterodimers, the dilutions of which weredetermined in titration experiments that resulted in substantialreduction of OD values after a 2-fold dilution. For the CD4 competitionassays, increasing concentrations of soluble or free CD4 ranging inconcentration from 10⁻¹¹ M up to 10⁻⁶ M diluted in 0.5% BSA/0.025% Tween20/PBS were admixed with soluble heterodimers. The plates weremaintained for 90-120 minutes at 37° C. and carefully washed ten timeswith 0.05% Tween 20/PBS before admixture of alkalinephosphatase-labelled goat anti-human IgG F(ab')2 at a dilution of 1:500followed by maintenance for 1 hour at 37° C. Development was performedas described for ELISA.

To establish the relationship between neutralizing ability as describedin Example 3 below could be related to antigen binding affinity ofHIV-1-specific Fabs, competition ELISAs were carried out where solublegp120 was competed with gp120 coated on ELISA plates for Fab binding.FIG. 7 shows that all Fabs were competed from binding to gp120 with aIC₅₀ of approximately 10⁻⁹ M free gp120. In addition as shown in Example3, there is no correlation between antigen affinity and neutralization.The Fabs tested included Fabs 4, 12, 21 and 7 that are members of thesame groups as determined by sequence analysis and comparison asdescribed in Example 9. Fabs 13, 27, 6, 29, 2 and 3 are all members ofthe different groups as determined by sequence analysis and comparisonas described in Example 9. Loop 2 is an Fab fragment selected from thesame library as the other Fabs but which recognizes the V3 loop. Onlywith the V3 loop peptide was competition carried out with gp120 from theSF2 strain.

To investigate whether neutralization could be associated with blockingof the gp120-CD4 interaction, competition ELISAs were carried out withsoluble CD4 competing with Fabs for binding to gp120-coated ELISA wells.The results are shown in FIG. 8. P4D10 and loop 2 are controls notexpected to be competed by CD4. P4D10 is a mouse monoclonal antibodyreacting with the V3 loop of gp120 (IIIB). Loop 2 Fab competition wascarried out using gp120 (SF2). As shown in FIG. 8 the binding of allFabs with the exception of the controls was inhibited with an IC₅₀ ofapproximately 10⁻⁸ M of soluble CD4. In addition, no difference wasdetected between the neutralizing and non-neutralizing Fabs to gp120inhibited by CD4. This implies that blocking of the CD4-gp120interaction is unlikely to be an important factor in Fab neutralizationof the HIV-1 virus.

Similar competition assays were performed with the Fabs panned againstthe four HIV-1 derived antigens. The 19 Fabs derived from panningagainst gp120 (IIIB) showed apparent affinities (1/concentration at 50%inhibition) for gp120 (IIIB) in the range 10⁷ -10⁻⁹ M with most being1-3×10⁻⁸ M. The panning procedure tends to select strongly for tightbinders so a grouping into a relatively narrow band of affinities wasexpected. Of 16 Fabs derived from panning against gp160 (IIIB), 6 werealso reactive with gp120 (IIIB) and competition ELISAs showed they hadsimilar apparent affinities as the gp120-panned Fabs. The non-gp120reactive clones from the gp160 panning showed a lower ELISA reactivitywith gp160 and could not be satisfactorily competed with gp160. They maybe directed against gp41 but were not pursued here. Eight Fabs derivedfrom panning against gp120 (SF2) also showed strong ELISA reactivitywith gp120 (IIIB) and gave similar apparent binding affinities. FourFabs were derived from panning against the V3 loop peptide. Of theseFabs, 2 reacted in ELISA with gp120 (SF2) but none with gp120 (IIIB).The apparent binding affinity of these loop binders to gp120 (SF2) was10⁻⁸ M.

To complete the survey in terms of strain cross-reactivity of Fabs,those derived from the gp120 and gp160 (IIIB) pannings were examined forELISA reactivity with gp120 (SF2). All were reactive. Therefore, all theFabs examined, with the exception of those selected by panning againstthe V3 loop peptide, bound to gp120 from IIIB and SF2 strains.

The Fabs were screened for CD4 inhibition of their binding to gp120(IIIB) immobilized on ELISA wells. All, again with the exception of theV3 loop binders, showed sensitivity to CD4 inhibition. The inhibitionconstants were in the range 10⁻⁷ to 10⁻⁹ M.

c) Binding Affinity Determination Using Surface Plasmon Resonance

Binding affinities were determined for six of the Fabs using surfaceplasmon resonance. Surface plasmon resonance was performed as it is amore accurate method for measuring affinity than competition ELISA. Thesix Fabs were chosen based upon sequence analysis which revealed thatthe heavy chains could be organized into 7 groups (Example 9). Eachgroup contained members with identical V-D and D-J joining regions,implying a common clonal origin with varying numbers of differenceselsewhere in the VH domain. Six Fabs were chosen as a representative ofeach respective group for further study as described herein. The singlemember of the seventh group was not included in these studies. Thebinding affinities of the six Fabs that are directed against the CD4binding site of the gp120 envelope glycoprotein were determined usingsurface plasmon resonance as follows.

A Pharmacia BIAcore machine was used for the binding affinitydeterminations as previously described in Malmborg, et al., J. Immunol.,35:643-650 (1992) and Mattsson, et al., J. Immunol. Meth., 145:229-240(1991). Optimization for the Fab fragments involved a number of steps.Two separate channels on a biosensor chip were coated with gp120 derivedfrom the HIV-1 strain LAI (Repligen, Cambridge, Mass.) such that onechannel could be used for the determination of on-rate constants(k_(on)) and the other for the determination of off-rate constants(k_(off)).

For immobilization of antigen on the sensor surfaces, a flow rate of 5μl/min of PBS, pH 7.4 was established over the biosensor chip. The chipwas then activated by injecting 30 μl of activation solution (PharmaciaBiosensor, 50% 0.2M N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide, 50%N-hydroxysuccinimide). The flow rate was then adjusted to 10 μl/min andthe gp120 was injected in 10 mM sodium acetate buffer, pH 4.5. Whenassociation rates were to be determined, 25 μl of gp120 at 10 μg/ml wasinjected (a final level of 4000 Response Units (RU)). Twenty μl of gp120at 2 μg/ml were injected for the determination of dissociation constants(a final level of 800 RU). In both cases, a flow rate of 5 μl/min wasreestablished following the gp120 injection and the chip was blockedfrom any further immobilization by the injection of 30 μl of 1Methanolamine, pH 8.5 (Pharmacia Biosensor).

For determination of on-rate constants (k_(on)), a series of dilutionswere made for each Fab to give final concentrations in the range of 1 to20 μg/ml. 30 μl of each Fab solution was injected in separateexperiments over the immobilized gp120 at a flow rate of 5 μl/min. Thechange in response per unit time (dR/dr) was plotted against time (t)for each concentration. The slopes of each of these graphs were thenplotted against their corresponding concentrations to give a final graphfrom which the on-rate constant could be read.

For determination of off-rate constants (k_(off)), 30 μl of each Fabsolution at 150 μg/ml were injected over the immobilized antigen at aflow rate of 5 μl/min. Once the reaction had reached equilibrium, theFab was removed from the antigen at a constant flow rate of 50 μl/min. Aplot was then made of ln(R_(i) /R₀) against t_(i) -t₀ for thedissociation phase. R_(i) is the response at time t_(i) and R₀ is theinitial response at time t₀. The slope of this graph was taken to be theoff-rate constant. Affinities (K_(a)) were then calculated and expressedas k_(on) /k_(off).

The apparent affinities of the panel of recombinant Fabs isolated fromthe donor as determined in competition ELISA and surface plasmonresonance were compared. Values of approximately 10⁸ M⁻¹ were obtainedby competition ELISA as described in Example 2b6c in which the solubleand immobilized gp120 competed for binding to Fab in bacterialsupernatants. Such a methodology only gives an approximate measure ofaffinity. Therefore, the affinities of six of these Fabs were measuredusing real-time biospecific interaction analysis (surface plasmonresonance) in order to obtain more accurate affinity constant values.The results are reproducible with a standard deviation from the mean ofapproximately 5% as determined by calculating a number of the affinityconstants in triplicate. All Fabs examined have affinities in the rangeof 5×10⁷ to 1 ×10⁸ M⁻¹ as determined in surface plasmon resonance (Table4). These values are in broad agreement with those derived fromcompetition ELISA. These values imply no correlation between affinityfor recombinant gp120 derived from LAI and the ability to neutralize theHXBc2 clone of HIV-1 derived from LAI as assessed in Example 3c.

                  TABLE 4    ______________________________________    Fab    k.sub.on (M.sup.-1 s.sup.-1)                          k.sub.off (s.sup.-1)                                   K.sub.a(M-1)    ______________________________________    b3     9.6 × 10.sup.3                          1.8 × 10.sup.-4                                   5.1 × 10.sup.7    b6     1.6 × 10.sup.4                          1.6 × 10.sup.-4                                   9.7 × 10.sup.7    b11    5.6 × 10.sup.4                          4.3 × 10.sup.-4                                   1.3 × 10.sup.8    b12    4.5 × 10.sup.4                          4.3 × 10.sup.-4                                   1.1 × 10.sup.8    b13    1.1 × 10.sup.4                          1.4 × 10.sup.-4                                   7.9 × 10.sup.7    b14    6.0 × 10.sup.4                          6.5 × 10.sup.-4                                   9.2 × 10.sup.7    ______________________________________

Also contemplated are competition ELISA and surface plasmon resonanceassays where the binding of HIV-1 recombinant Fabs of this invention isperformed in the presence of excess Fabs of this invention as well asthose HIV-1 antibodies, polyclonal or monoclonal, present in patientsera, either asymptomatic or symptomatic, or obtained by other meanssuch as EBV transformation and the like. The ability of an exogenouslyadmixed antibody to compete for the binding of a characterized Fab ofthis invention will allow for the determination of equivalent antibodiesin addition to unique epitopes and binding specificities.

3. Neutralizing Activity of Recombinant Human Fab Fragments AgainstHIV-1 In Vitro

Binding of antibodies to viruses can result in loss of infectivity orneutralization and, although not the only defense mechanism againstviruses, it is widely accepted that antibodies have an important role toplay. However, understanding of the molecular principles underlyingantibody neutralization is limited and lags behind that of the othereffector functions of antibody. Such understanding is required for therational design of vaccines and for the most effective use of passiveantibody for prophylaxis or therapy. This is particularly urgent for thehuman immunodeficiency viruses.

A number of studies have led to the general conclusion that viruses areneutralized by more than one mechanism and the one employed will dependon factors such as the nature of the virus, the epitope recognized, theisotype of the antibody, the cell receptor used for viral entry and thevirus:antibody ratio. The principle mechanisms of neutralization can beconsidered as aggregation of virions, inhibition of attachment of virusto cell receptor and inhibition of events following attachment such asfusion of viral and cellular membranes and secondary uncoating of thevirion. One of the important features of the third mechanism is that itmay require far less than the approximately stoichiometric amounts ofantibody expected for the first two mechanisms since occupation of asmall number of critical sites on the virion may be sufficient forneutralization. For instance it has been shown that neutralization ofthe influenza A virion obeys single hit kinetics as described by Outlawet al., Epidemiol. Infect., 106:205-220 (1992).

Intensive studies have been carried out on antibody neutralization ofHIV-1. For review, see Nara et al., FASEB J., 5:2437-2455 (1991). Mosthave focussed on a single linear epitope in the third hypervariabledomain of the viral envelope glycoprotein gp120 known as the V3 loop.Antibodies to this loop are suggested to neutralize by inhibiting fusionof viral and cell membranes. Binding to the loop resulting inneutralization can occur prior to virus-cell interaction or followinggp120 binding to CD4. See, Nara, In Retroviruses of Human Aids andRelated Animal Diseases, eds. Girard et al., pp. 138-150 (1988); Linselyet al., J. Virol., 62:3695-3702 (1988); and Skinner et al., J. Virol.,67:4195-4200 (1988). Features of the V3 loop are sequence variabilitywithin the loop [Goudsmit et al., FASEB J., 5:2427-2436 (1991) andAlbert et al., AIDS, 4:107-112 (1990)] and sensitivity of neutralizingantibodies against the loop to sequence variations outside the loop[Nara et al., FASEB J., 5:2437-2455 (1991); Albert et al., supra;McKeating et al., AIDS, 3:777-784 (1989); and Wahlberg et al., AIDS Res.Hum. Retroviruses, 7:983-990 (1991). Hence anti-V3 loop antibodies areoften strain specific and mutations in the loop in vivo may provide amechanism for viral escape from antibody neutralization.

Recently considerable interest has focused on antibodies capable ofblocking CD4 binding to gp120. A number of groups have described thefeatures of these antibodies as (a) reacting with conformational i.e.,non-linear epitopes, (b) reacting with a wide range of virus isolatesand (c) being the predominant neutralizing antibodies in humans afterlonger periods of infection. See, Berkower, et al., J. Virol.,65:5983-5990 (1991); Steimer et al., Science, 254:105-108 (1991); Ho etal., J. Virol., 65:489-493 (1991); Kang et al., Proc. Natl. Acad. Sci.,USA, 88:6171-6175 (1991); Posner et al., J. Immunol., 146:4325-4332(1991); and Tilley et al., Res. Virol., 142:247-259 (1991). Neutralizingantibodies of this type would appear to present a promising target forpotential therapeutics. The mechanism(s) of neutralization of theseantibodies is unknown although there is some indication that this maynot be blocking of virus attachment since a number of mouse monoclonalantibodies inhibiting CD4 binding to gp120 are either non-neutralizingor only weakly neutralizing.

The generation of human monoclonal antibodies against the envelope ofHIV-1 as described by Burton et al., Proc. Natl. Acad. Sci., USA,88:10134-10137 (1991) using combinatorial libraries allows a novelapproach to the problem of neutralization. Given the lack of athree-dimensional structure for gp120 and the complexity of the virus,the approach seeks to explore neutralization at the molecular levelthrough the behavior of related antibodies. This is possible for thefollowing reasons: (1) the combinatorial approach allows the rapidgeneration of large numbers of human antibodies; (2) the antibodies (Fabfragments) are expressed in E. coli and can readily be sequenced; and(3) antibodies have similar sequences and common structural motifsallowing functional differences to be meaningfully correlated withprimary structure.

Neutralization studies were performed as described herein on the humanrecombinant Fab fragments from 20 clones against gp120 prepared asdescribed in Examples 1 and 2, all of which are strain cross-reactiveand inhibited by CD4 from binding to gp120. The results presented hereinshow that neutralization was not effected by virus aggregation orcross-linking of gp120 molecules on the virion surface and was notcorrelated with blocking of the interaction between soluble CD4 andrecombinant gp120.

Neutralization studies were also performed as described herein on thehuman recombinant Fab fragments from the gp41-reactive clones preparedas described in Examples 1 and 2. The results are presented below.

Two different assays, a p24 ELISA assay and a syncytium assay, wereperformed to measure neutralization ability of the recombinant humanHIV-1 immunoreactive Fabs. An additional assay, a plaque assay, wasperformed for determining the neutralization effectiveness of thegp41-reactive Fabs. In plaque assays, CD4+ cells were cultured in thepresence or absence of soluble gp41-reactive Fabs prior to inoculationwith virus. Inhibition of infectivity, also referred to asneutralization, by antibodies was expressed as the percent of plaqueformation in the cultures compared to cells exposed to PBS alone.

Neutralization assays were also performed with an antibody moleculeconsisting of the light chain and the VH region of the Fab 12 and theconstant regions (CH1, CH2, and CH3) of an IgG1 molecule. Quantitativeinfectivity microplaque and syncytial formation assays to measureneutralization were performed with the b12 IgG1 and laboratory isolatesMN and IIIB of HIV-1 virus. In the syncytial formation assay, virus wasgrown in H9 cells and infectivity measured by culturing monolayers ofCEM-SS target cells with 100-200 syncytial forming units (SFUs) ofvirus, in the presence or absence of antibody. p24 ELISA and microplaqueformation assays were also performed with primary clinical isolates ofthe HIV-1 virus.

In addition, the ability of the recombinant human HIV-1 immunoreactiveFabs b3, b6, b12, b13, and b12 to neutralize the HXBc2 molecular cloneof gp120 derived from HTLV-IIIB (LAI) was determined in an envelopecomplementation assay. The supernatant containing recombinant HIV-1virions from cotransfected COS-1 cells was incubated with therecombinant Fabs prior to incubation with Jurkat cells. The recombinantHIV-1 virions contained the HXBc2 clone of HIV-1 strain LAI whichencodes a chloramphenicol acetyltransferase (CAT) gene. Upon infectionof Jurkat cells with the recombinant HIV-1 virions, the CAT gene wasexpressed and CAT activity measured. Activity of the CAT gene wastherefore an indication of infectivity of the Jurkat cells with therecombinant HIV-1 virion. Lack of CAT activity indicated the Jurkatcells were not infected with the recombinant HIV-1 virion.

For some of these assays, the recombinant Fabs were first purified. Oneliter cultures of SB containing 50 μg/ml carbenicillin and 20 mM MgCl₂were inoculated with appropriate clones and induced 7 hours later with 2mM IPTG and grown overnight at 30° C. The cell pellets were sonicatedand the resultant supernatant were concentrated to a 50 ml volume. Thefiltered supernatants were loaded on a 25 ml protein G-anti-Fab column,washed with 120 ml buffer at a rate of 3 ml/minute and eluted withcitric acid at pH 2.3. The neutralized fractions were then concentratedand exchanged into 50 mM MES at pH 6.0 and loaded onto a 2 ml Mono-Scolumn at a rate of 1 ml/minute. A gradient of 0-500 mM NaCl was run at1 ml/minute with the Fab eluting in the range of 200-250 mM NaCl. Afterconcentrating, the Fabs were positive when titered on ELISA againstgp120 and gave a single band at 50 kD by 10-15% SDS-PAGE. Concentrationwas determined by absorbance measurement at 280 nm using an extinctioncoefficient (1 mg/ml) of 1.4.

a. Neutralization as Measured by the p24 ELISA Assay

For this assay, diluted tissue culture supernatants of HIV-1 IIIB orMN-infected peripheral blood mononuclear cells (PBMC) (50TCID₅₀ (50%tissue culture infectious dose), 100 μl) were maintained for 2 hours at37° C. with serial dilutions (1:2), beginning at a dilution of 1:20, ofrecombinant Fab supernates prepared in Example 266). Control Fabsupernates were also provided that included human neutralizing sera, aknown human neutralizing monoclonal antibody 2F5 and the Fab fragmentderived from that antibody by papain digestion, and a known mouseneutralizing monoclonal antibody and its F(ab')₂ fragment as describedby Broliden et al., J. Virol., 64:936-940 (1990). PBMC (1×10⁵ cells )were admixed to the virus/antibody admixture and maintained for 1 hourat 37° C. Thereafter, the cells were washed and maintained in RPMI 1640medium (GIBCO) supplemented with 10% fetal calf serum, 1% glutamine,antibiotics and IL-2. The culture medium was changed at days 1 and 4. At7 days post-infection, supernates were collected and analyzed by HIV-1p24 antigen capture ELISA as described by Sundqvist et al., J. Med.Virol., 29:170-175 (1989) the disclosure of which is hereby incorporatedby reference. Neutralization was defined as positive if an 80% orgreater reduction of optical density at 490 nm in the culturesupernatant occurred as compared to negative Fab or negative humanserum. Tests with all Fabs, mAbs and sera were repeated on at least twooccasions.

b. Quantitative Infectivity Assay Based on Syncytial Formation

A quantitative neutralization assay with the MN strain of HIV-1 wasperformed as described by Nara et al., AIDS Res. Human Retroviruses,3:283-302 (1987), the disclosure of which is hereby incorporated byreference. Monolayers of CEM-SS target cells were cultured with virus,in the presence or absence of antibody, and the number of syncytiaforming units determined 3-5 days later. An equivalent amount of viruswas used in the assays to allow direct comparison of the variousantibody concentrations tested. The assays were repeatable over avirus-surviving fraction range of 1 to 0.001 within a 2 to 4-folddifference in the concentration of antibody (P<0.001).

c. Neutralization as Measured by the Envelope Complementation Assay

The ability of purified recombinant Fabs b3, b6, b11, b12, b13, and b14to neutralize the HXBc2 gp120 molecular clone of the HIV-1 (LAI) isolatewas assessed in an envelope complementation assay (Helseth et al., J.Virol., 65:2119-2123 (1991)). Briefly, COS-1 cells were cotransfectedwith a plasmid expressing envelope glycoprotein 120 derived from HIV-1(LAI) and a plasmid containing an env-defective HXBc2 clone and encodingthe bacterial CAT gene. Equal fractions of the cell supernatantscontaining recombinant virions were incubated at 37° C. for 1 hour withvarying concentrations of recombinant Fab (0.1-20 μg/ml) or controlmonoclonal antibody 110.4 prior to incubation with Jurkat cells. Threedays post-infection, the Jurkat cells were lysed and CAT activitymeasured. Neutralization was expressed as a decrease in the percentageof residual chloramphenicol transferase (CAT) activity. Controlmonoclonal antibody 110.4 is a strongly neutralizing antibody directedto the V3 loop of the HXBc2 HIV-1 strain.

d. Results of the Neutralization Assays for gp120

Assays were generally repeated at least twice with reproducible results.For the data reported in FIG. 6, the gp120-specific Fab supernates weredivided into two parts, one being used in the p24 assay and the other inthe syncytia assay. A dash (--) indicates that there was noneutralization at 1:20 dilution in the p24 assay and 1:16 in thesyncytial assay (with most clones showing no detectable neutralizationat a 1:4 dilution). Neutralization titers are indicated in the figure.For the p24 assay, the titer corresponds to the greatest dilutionproducing >80% reduction in absorbance in ELISA. For the syncytia assay,Fabs 4 and 12 produced >95% neutralization at a 1:4 dilution ofsupernate and 80 and 70% reduction at 1:128 dilution respectively. TheseFabs were effective neutralizers in both types of assays. They have alsobeen shown to neutralize infection by IIIB and RF strains using aPCR-based assay of proviral integration. Fabs 6 and 7 showed noneutralization in the syncytia assay but other supernate preparationsshowed activity. Fab 13 was consistently effective in the p24 assay butnot in the syncytia assay. A number of other clones show lower levels ofneutralizing ability.

Fabs were purified from a selection of some of the clones as describedabove and used in both neutralization assays. As shown in FIG. 9, Fabs 4and 12 were again effective in both assays at similar levels with forexample 50% inhibition of syncytial formation at an Fab concentration ofapproximately 20 nM (1 μg/ml). The results shown are derived from thesyncytia assay using the MN strain. Fabs 7 and 21 were equally effectivein the syncytial assay but somewhat less so in the p24 assay. The p24assay indicated greater than 80% neutralization of HIV-1MN strain forFab 4 at 3, Fab 7 at 15, Fab 12 at 3, Fab 13 at 4 and Fab 21 at 7 μg/ml,respectively. Fab 13 however was ineffective in the syncytial assay at25 μg/ml. For the IIIB strain, greater than 80% neutralization wasobserved for Fab 4 at 13, Fab 7 at 15, Fab 12 at 7 and Fab 21 at 14μg/ml, respectively. Although Fab 11 was not effective in neutralizationassays when unpurified as shown in FIG. 6, following purification, Fab11 was equally effective as Fab 12 in neutralizing HIV-1. For thisreason, the Fab is being deposited with the ATCC as described in Example12 along with Fab 12 and Fab 13.

The ability of purified recombinant Fabs b3, b6, b11, b12, b13, and b14to neutralize the HXBc2 gp120 molecular clone of the HIV-1 (LAI) isolatewas assessed in an envelope complementation assay. FIG. 23 shows theconcentration dependence of Fab neutralization of the HXBc2 clone inthis assay. All of the Fabs neutralize effectively at the highestconcentration measured (20 μg/ml). Irrelevant Fabs, Fabs directed tosurface glycoproteins on other viruses such as RSV, do not neutralize inthis assay. Examination of the lower concentrations clearly reveals thatFab b12 is the most effective neutralizer. The neutralizing potency ofFab b12 was greater than that of the 110.4 whole monoclonal antibodytested in parallel. The 110.4 antibody is one of the most potentantibodies directed against the V3 loop of the HXBc2 HIV-1 strain(Thali, M. and J. Sodroski, unpublished observations). In other studies,Fab b12 has been found to show exceptional neutralizing ability towardslaboratory (Example 3 and Barbas et al., Proc. Natl. Acad. Sci., USA,91, in press (1994)) and field isolates of HIV-1 as described in Example5.

There are a number of conclusions arising from the data shown in theFIGS. 6, 9 and 23. It is apparent that HIV-1 can be neutralized withoutvirion aggregation or cross-linking of gp120 molecules on the virionsurface since monovalent Fab fragments are effective. To further confirmthis finding, a Fab fragment was produced by papain digestion of a knownneutralizing human monoclonal antibody. As shown in FIG. 6, the Fabfragment was approximately equally effective as the whole IgG inneutralization of the MN strain of HIV-1. This is consistent withresults on Fabs prepared from two mouse monoclonal antibodies to the V3loop. An F(ab')₂ fragment of a mouse monoclonal antibody was somewhatless effective than the parent IgG in neutralization of the MN strain.interestingly, the fragments from these control antibodies wererelatively poor in neutralizing the IIIB strain of HIV-1. The resultsalso show that there appears to be a difference between the two assaysemployed since Fab 13 was consistently effective in one assay but notthe other. The principal variables were the incubation time of the virusand antibody prior to infection (2 hours for the p24 assay and 0.5 hoursfor the syncytial assay), the amount of virus used for infection, thecells used to propagate virus (human PBMCs for the former and H9 cellsfor the latter) and the cells infected (human PBMCs for the former andCEM.SS cells for the latter). Of these, there is a strong possibilitythat the MN virus used in the two assays, having been passaged throughdifferent cells, is critically different.

The Fabs show a spectrum of neutralizing ability for gp120 from amolecular clone HXBc2 derived from the HIV-1 strain LAI in the envelopecomplementation assay. Fab b12 exhibited the greatest potency ofneutralization and was even more effective in this assay than a wholeantibody directed to the V3 loop of gp120. Neutralizing ability is notcorrelated with either the apparent affinity of the Fab for gp120derived from the recombinant HIV-1 strain LAI as estimated bycompetition ELISA or the affinity for gp120 derived from HIV-1 strainLAI as determined by surface plasmon resonance. For example, Fabs b6,b12, and b14 have very similar affinities by surface plasmon resonance(Table 4) but different neutralization ability in the envelopecomplementation assay (FIG. 23). Similarly, neutralization is notcorrelated with the ability of the Fab to compete with soluble CD4 in acompetition ELISA.

e. Results of the Neutralization Assays for gp41

The gp41-reactive Fabs exhibited specificity to the conformation epitopeof gp41 including amino acid residues in positions 565-585 and 644-663.The five selected gp41-specific Fabs were designated DL 41 19, DO 41 11,GL 41 1,MT 41 12 and SS 41 8. Neutralization assays were performed asdescribed above for the gp120-reactive Fabs. In the plaque assays, thedata shown is the concentration of Fab in micrograms/milliliter requiredto achieve 50% of neutralization. The data for the other twoneutralization assays is also expressed in micrograms/milliliter of Fabrequired to neutralize infection as defined in the description of thep24 and syncytial assays above. The results of the three neutralizationassays, plaque, syncytial and p24, are presented in Table 5. The MN andIIIB HIV strains were used as indicated in Table 5 for the assays. Theabbreviation "ND" stands for not determined when indicated in the table.

                  TABLE 5    ______________________________________    Assay/Strain            Plaque   Syncytial  P24    Fab      MN       IIIB   IIIB     MN   IIIB    ______________________________________    DL 41 19 <4       <40    1.4      ND   ND    DO 41 11 <40      7.1    2.3      0.9  ND    GL 41 1  <4       <4     1.7      ND   3.5    MT 41 12 <40      <40    5.5      4.5  4.5    SS 41 8  <4       <4     2.2      ND   7.1    ______________________________________

As shown in Table 5, all five Fabs were effective at neutralizing bothMN and IIIB strains of HIV in either plaque, syncytial or p24 assays.Fabs DL 41 19 and DO 41 11 exhibited strain specificity in the plaqueassay where the former was ten-fold more effective at inhibiting plaqueformation with the MN strain than with the IIIB strain. The oppositespecificity was seen with the DO 41 11 Fab. However, both Fabs exhibitedcomparable neutralization as measured by the syncytial assay. Two Fabs,GL 41 1 and SS 41 8, were equally effective at inhibiting plaqueformation with either MN or IIIB strains. The Fab MT 41 12 was similarlynot strain-specific although neutralization required 10 fold moreantibody. No strain specificity was evident when Fab MT 41 12 was usedin p24 assays where the same amount of antibody was equally effective.All five antibodies were neutralized IIIB as measured in the syncytialassay.

Thus, the five gp41-specific Fabs neutralized HIV-1MN and IIIB in atleast two of the three assays used for measuring neutralizing activity.Moreover, strain specificity was prevalent in two of the five assays asmeasured by the plaque assay. Based on these differential neutralizationcharacteristics, the gp41-specific Fabs provide useful therapeuticreagents for neutralizing HIV-1.

4. Construction of a Mammalian Expression Vector pEe12 Combo BM 12 forthe Expression of an IgG1 Antibody Molecule with the Fab from b12 (b12IgG1)

Although Fab b12 is capable of neutralizing some primary isolates, thecorresponding whole antibody molecule is likely to be more effective.The whole antibody, consisting of the Fab fragment and the Fc domain,participates in the elimination of foreign cells by first bindingspecifically to the foreign cell via the Fab portion and interactingwith other cells in the immune system via the Fc domain. The Fc domainalso enables the antibody to bind complement.

Fab b12 was converted to a whole IgG1 molecule (b12 IgG1) by cassettingthe variable heavy chain (VH) and light chain genes into a vectorcreated for high-level mammalian expression. b12 IgG1 used in theneutralization studies was prepared by expression in Chinese hamsterovary (CHO) cells and purified by affinity chromatography.

The strategy to convert the Fab b12 to a whole IgG1 molecule was similarto that described previously for the generation of a whole antibodybeginning with a phage derived Fab (Bender, et al., Hum. Antibod.Hybridomas, 4:74-79 (1992)).

a. Construction of b12 Heavy Chain IgG1 pSG-5 Mammalian ExpressionVector

1) Modification of b12 Heavy Chain Variable Region to Introduce a KozakSequence, Mammalian Leader Sequence, and Human VH Consensus Sequence

First, the b12 VH region was cloned into a pSG-5 expression vector(Green et al., Nucl. Acids Res., 16:369 (1988)) to fuse the b12 VH tothe heavy chain constant domains (CH1, CH2, and CH3) of an IgG1 antibodymolecule. The double-stranded Fab b12 DNA was used as a template forisolating the gene encoding the VH region of the Fab b12, the amino acidresidue sequence of which is listed in SEQ ID NO 66. Fab b12 DNA andmouse B73.2 IgG1 DNA (Whittle, et al., Protein Eng., 1:499 (1987) andBruggmeman, et al., J. Exp. Med., 166:1351 (1987)) were used astemplates for a PCR amplification for the construction of a DNA fragmentconsisting of the unique Kozak sequence for the control of heavy chainexpression, the mouse B72.3 heavy chain leader sequence(MEWSWVFLFFLSVTTGVHS (SEQ ID NO 155 from amino acid residue sequence 1to 20)), the human VH consensus sequence (QVQLVQ (SEQ ID NO 155 fromamino acid residue sequence 21 to to 26)), and the VH region of the Fabb12. Altering the beginning of the VH from the mouse consensus sequenceto the human consensus sequence also destroyed the original Xho Icloning site. The restriction sites EcoR I and Sst I were introduced inthe amplification reaction and were located at the 5' and 3' ends of thefragment, respectively. The procedure for creating the modified VHfragment by combining the products of the two separate PCRamplifications is described below.

The primer pair, HC-1 (SEQ ID NO 157) and HC-2 (SEQ ID NO 158) as shownin Table 10, was used in the first PCR reaction to amplify a portion ofthe Fab b12 VH gene and incorporate the human heavy chain consensussequence into the 5' end of the VH fragment and introduce an Sst Icloning site in the 3' end of the VH fragment. In addition, the 5' PCRprimer introduces sequences into the VH fragment which form 27 basepairs of homology with the mouse leader sequence fragment preparedbelow. The 27 base pairs of homology in the fragments is used in asubsequent PCR reaction to fuse the two PCR products (Yon and Fried,Nucl. Acids Res., 17:4895 (1989)) to form a modified VH fragmentconsisting of the EcoR I cloning site, the mouse leader sequence 72.3,the human consensus sequence, the remaining VH coding sequence, and theSst I cloning site. For the PCR reactions, 1 μl containing 100 ng of Fabb12 DNA was admixed with 10 μl of 10× PCR buffer in a 0.5 ml microfugetube. To the DNA admixture, 8 μl of a 2.5 mM solution of dNTPs (dATP,dCTP, dGTP, dTTP) was admixed to result in a final concentration of 200micromolar (μM) of each dNTP. 1 μl (equivalent to 20 picomoles (pM)) ofthe 5' forward HC-1 primer and 1 μl (20 pM) of the 3' backward HC-2primer were admixed into the DNA solution. To the admixture, 73 μl ofsterile water and 2.5 units of Taq DNA polymerase was added. Two dropsof mineral oil were placed on top of the admixture and 35 rounds of PCRamplification in a thermocycler were performed. The amplification cycleconsisted of 52° C. for 1 minute, 72° C. for 2 minutes and 94° C. for0.5 minutes.

The primer pair, HC-3 (SEQ ID NO 159) and HC-4 (SEQ ID NO 160) as shownin Table 10, was used in a separate PCR reaction to amplify the mouseB72.3 leader sequence and incorporate an EcoR I cloning site at the 5'end of the fragment and to introduce a 27 base pair sequence which hashomology to the modified VH fragment prepared above. Double-stranded DNAencoding the mouse B73.2 IgG1 (Whittle, et al., supra) was used as atemplate for preparation of the mouse 72.3 leader sequence. The PCRreaction to prepare the mouse leader sequence fragment was performedusing the same conditions as described above for the preparation of themodified VH fragment.

The resultant PCR modified b12 VH DNA fragment and mouse leader sequencefragment were purified by electrophoresis in a 2.5% Nu-Sieve agarose gel(FMC). The area in the agarose containing the modified b12 VH DNAfragment and mouse leader sequence fragment were excised from theagarose.

A third PCR amplification using the primer pairs, HC-1 (SEQ ID NO 157)and HC-3 (SEQ ID NO 159) as shown in Table 10, was performed to fuse themouse leader fragment with the modified VH fragment. The primers usedfor this amplification were designed to preserve an EcoR I site, aunique Kozak sequence, and the mouse B72.3 heavy chain leader sequenceon the 5' end of the amplified fragment and to preserve the Sst Icloning site on the 5' end of the amplified fragment. The templates usedin this PCR reaction were the two purified PCR reaction productsdescribed above. The PCR reaction and subsequent purification of the PCRproduct were performed as described above.

2) Modification of b12 Heavy Chain Variable Region to Eliminate a BglIIRestriction Site

The b12 modified heavy chain fragment prepared in Example 4al containeda Bgl II cloning site at amino acid residue 87 which would interferewith the insertion of the heavy chain fragment into the pEE6 mammalianexpression vector. The Bgl II restriction site was therefore eliminatedin a PCR reaction using primers which destroyed the Bgl II restrictionsite while preserving the encoded amino acid, arginine at amino acidresidue 87 of the modified b12 heavy chain fragment.

The primer pair, HC-1 (SEQ ID NO 157) and HC-6 (SEQ ID NO-162) as shownin Table 10, was used in the first PCR reaction to preserve the 5'region of the modified b12 heavy chain fragment and destroy the IIrestriction site at amino acid residue 87 of the heavy chain. The HC-6primer introduces sequences into the VH fragment which form 32 basepairs of homology with the remaining portion of the VH fragment whichwill be prepared as described below. The 32 base pairs of homology inthe fragments was used in a subsequent PCR reaction to fuse the two PCRproducts (Yon and Fried, supra) to form a modified VH fragment asdescribed above but without the Bgl II restriction site. The PCRreaction was performed and the PCR products were purified as describedin Example 4a1.

The primer pair, HC-2 (SEQ ID NO 142) and HC-5 (SEQ ID NO 145) as shownin Table 10, was used in the second PCR reaction to preserve the 3'region of the modified b12 heavy chain fragment and destroy the IIrestriction site. The HC-5 primer introduces sequences into the VHfragment which form 32 base pairs of homology with the remaining portionof the VH fragment which was prepared in the first PCR reaction. PCRproducts which have incorporated the HC-5 and HC-6 primers contain 32base pairs of overlapping sequences which are identical. It is theannealing of the two PCR products at these 32 base pairs during thesubsequent PCR reaction which fuses the two portions of the VH fragmenttogether to recreate the entire VH fragment as described in Yon andFried (supra).

A third PCR amplification using the primer pairs, HC-1 (SEQ ID NO 157)and HC-3 (SEQ ID NO 159) as shown in Table 10, was performed to fuse thetwo VH fragments in which the Bgl II restriction site had beendestroyed. The primers used for this amplification were designed topreserve an EcoR I site, a unique Kozak sequence, and the mouse B72.3heavy chain leader sequence on the 5' end of the amplified fragment andthe Sst I cloning site on the 3' end of the amplified fragment. Thetemplates used in this PCR reaction were the two purified PCR reactionproducts described above. The PCR reaction and subsequent purificationof the PCR product were performed as described in Example 4a1.

                  TABLE 10    ______________________________________    SEQ    ID NO Primer    ______________________________________    (141).sup.1          HC-1     (F)   5' CAGGTTCAGCTGGTTCAGTCCGGGG                          CT 3'    (142).sup.2          HC-2     (B)   5' CCTTGGAGCTCACGATGACCGTGGT                          TCCTTGGCCCCAGACGTCC3'    (143).sup.3          HC-3     (F)   5' GGCCGCGAATTCGCCGCCACCATGG                          AATGGAGCTGGGTCTTTCTCTTCTT                          CCTGTCAGTA 3'    (144).sup.2          HC-4     (B)   5' AGCCCCGGACTGAACCAGCTGAAC                          CTG 3'    (145).sup.4          HC-5     (F)   5' GGAGTTGAGGAGCCTCAGGTCTGCA                          GACACGG 3'    (146).sup.4          HC-6     (B)   5' CCGTGTCTGCAGACCTGTGGCTCCT                          CAACTCC 3'    (147) LC-1     (F)   5' GATGCCAGATGTGAGATCGTTCTCA                          CGCAGTCT 3'    (148).sup.3,5          LC-2     (B)   5' GCGGGATCCGAATTCTCTAGAATTA                          ACACTCTCCCCTGTTGAAGCTCTTT                          GTGACGGGCGAACTCAG 3'    (149).sup.3          LC-3     (F)   5' GCGCGAATTCACCATGGGTGTGCCC                          ACTCAGGTCCTGGGGTTGCTGCTGC                          3'    (150) LC-4     (B)   5' AGACTGCGTGAGAACGATCTCACAT                          CTGGCATC 3'    (151).sup.6          LC-5     (F)   5' GCGCAAGCTTACCATGGGTGTGCCC                          ACTCAGGTCCTGGGGTTGCTGCTGC                          3'    ______________________________________     F Forward Primer     B Backward Primer     .sup.1 the Sst I cloning site is single underlined     .sup.2 the primers, HC2 and HC4 contain complementary sequences     .sup.3 the EcoR I cloning site is single underlined     .sup.4 in HC4, the G that is double underlined was altered from an A to     eliminate a Bgl II restriction site; in HC5, the C that is     doubleunderlined was altered from a T to eliminate a Bgl II restriction     site     .sup.5 the base A that is double underlined was introduced in the PCR     primer to alter the encoded amino acid from an arginine, R, to a serine,     .sup.6 the HindIII cloning site is single underlined

3) Insertion of Modified b12 Heavy Chain Variable Region into the pSG-5Mammalian Expression Vector

The modified b12 heavy chain variable region PCR product was ligatedinto a mammalian expression vector (Adair, et al., Hum. Antibod.Hybridomas, in press). The mammalian expression vector consisted of thepSG-5 vector (FIG. 24) with a human IgG1 gene inserted at the EcoR Isite. The human IgG1 gene contained a VH insert in the same readingframe as the constant regions of the human IgG1 gene. The VH insert wasremoved by digestion with EcoR I and Sst I enzymes. The constant regions(CH1, CH2, and CH3) remained in the pSG-5 vector. Transcription of theheavy chain gene in the pSG-5 expression vector is under the control ofthe SV40 early promoter. Transcriptional termination is signaled by theSV40 polyadenylation signal sequence downstream of the heavy chainsequence. The M13 intergenic region allows for the production ofsingle-stranded DNA for nucleotide sequence determination.

The modified b12 heavy chain variable region PCR product was digestedwith EcoR I and Sst I and purified on a 2.5% Nu-Sieve agarose gel (FMC).The mammalian expression vector DNA containing the IgG1 sequences wasdigested in parallel with EcoR I and Sst I enzymes to remove theoriginal VH region. The PCR modified heavy chain variable region wasligated to the constant regions in the mammalian expression vector usingT4 DNA ligase under conditions well known to those of skill in the artand transformed into DH5α competent cells following the manufacturer'srecommended procedures (GIBCO, BRL Life Technologies, Gaithersburg,Md.). The PCR modified heavy chain variable region was inserted in thesame reading frame as the constant regions of the human IgG1 gene in thepSG-5 vector. Miniprep DNAs were analyzed and large scale plasmidpreparations performed. The nucleotide sequence of the 5' untranslatedregion including the Kozak sequence, mouse B72.3 heavy chain leadersequence, heavy chain variable region, heavy chain constant regions, andSV40 signal sequence was determined by the dideoxy-nucleotide chaintermination method (Sanger et al., supra).

b. Construction of a b12 Light Chain pSG-5 Mammalian Expression Vector

1) Modification of b12 Light Chain to Introduce a Kozak Sequence,Mammalian Leader Sequence, and Human Light Chain Consensus Sequence

The b12 light chain was cloned into a separate pSG-5 expression vector(Green et al., supra). The double-stranded Fab b12 DNA was used as atemplate for isolating the gene encoding the light chain of the Fab b12,the amino acid residue sequence the light chain of Fab b12 is listed inSEQ ID NO 97. Mouse B73.2 IgG1 DNA (Whittle, et al., Protein Eng., 1:499(1987) and Bruggmeman, et al., J. Exp. Med., 166:1351 (1987)) was usedas a template for isolating the mouse B73.2 leader sequence. Fab b12 andmouse B73.2 IgG1 DNA were thus used as templates for a PCR amplificationfor the construction of a DNA fragment consisting of the unique Kozaksequence for control of light chain expression, the mouse B72.3 lightchain leader sequence (MGVPTQLGLLLWLTDARC (SEQ ID NO 153 from amio acidresidue sequence 1 to 20)), and the b12 light chain beginning with ahuman light chain amino acid consensus sequence (EIVLTQSP (SEQ ID NO 153from amino acid residue sequence 21 to 28)). Altering the beginning ofthe light chain from the mouse amino acid consensus sequence to thehuman amino acid consensus sequence also destroys the original Sac Icloning site. The restriction site, EcoR I, was introduced in theamplification reactions and was located at both the 5' and 3' ends ofthe fragment. The procedure for creating this fragment by combining theproducts of two separate PCR amplifications is described below.

The primer pair, LC-1 (SEQ ID NO 163) and LC-2 (SEQ ID NO 164), was usedin the first PCR reaction as performed above to amplify the Fab b12light chain gene and incorporate the human light chain consensussequence into the fragment and the EcoR I cloning site into the 3' endof the b12 light chain fragment. For the PCR reaction, 1 μl containing100 ng of Fab b12 DNA was admixed with 10 μl of 10× PCR buffer in a 0.5ml microfuge tube. To the DNA admixture, 8 μl of a 2.5 mM solution ofdNTPs (dATP, dCTP, dGTP, dTTP) was admixed to result in a finalconcentration of 200 μM of each dNTP. 1 μl (equivalent to 20 pM) of theLC-1 primer and 1 μl (20 pM) of the 3' backward LC-2 primer was admixedinto the DNA solution. To the admixture, 73 μl of sterile water and 2.5units of Taq DNA polymerase was added. Two drops of mineral oil wereplaced on top of the admixture and 35 rounds of PCR amplification in athermocycler were performed. The amplification cycle consisted of 52° C.for 1 minute, 72° C. for 2 minutes and 94° C. for 0.5 minutes.

The primer pair, LC-3 (SEQ ID NO-165) and LC-4 (SEQ ID NO 166) as shownin Table 10, was used in a separate PCR reaction to amplify the mouselight chain B72.3 leader sequence and incorporate an EcoR I cloning siteat the 5' end of the fragment and to introduce a 27 base pair sequencewhich has homology to the modified light chain fragment prepared above.Double-stranded DNA encoding the mouse B73.2 IgG1 (Whittle, et al.,supra) was used as a template for preparation of the mouse 72.3 leadersequence. The PCR reaction to prepare the mouse leader sequence fragmentwas performed using the same conditions as described in Example 4a forthe preparation of the modified VH fragment.

The resultant PCR modified b12 light chain DNA fragment and light chainmouse leader sequence fragment were purified by electrophoresis in a2.5% Nu-Sieve agarose gel (FMC). The area in the agarose containing themodified b12 light chain DNA fragment and light chain mouse leadersequence fragment were excised from the agarose.

A third PCR amplification using the primer pairs, LC-1 (SEQ ID NO 157)and LC-4 (SEQ ID NO 166) as shown in Table 10, was performed to fuse thelight chain mouse leader fragment with the modified light chainfragment. The primers used for this amplification were designed topreserve an EcoR I site, a unique Kozak sequence, and the mouse B72.3light chain leader sequence on the 5' end of the amplified fragment andto preserve the EcoR I cloning site on the 5' end of the amplifiedfragment. The templates used in this PCR reaction were the two purifiedPCR reaction products described above. The PCR reaction and subsequentpurification of the PCR product were performed as described in Example4a1.

2) Insertion of Modified b12 Light Chain into pSG-5 Mammalian ExpressionVector

The modified b12 light chain PCR product was ligated to a pSG-5 vector(FIG. 24). The pSG-5 vector had the same features described in Example4a2 but did not contain a human IgG1 gene.

The modified b12 light chain PCR product was digested with EcoR I andpurified on a 2.5% Nu-Sieve agarose gel (FMC). The pSG-5 vector DNA wasdigested in parallel with EcoR I enzyme. The PCR modified light chainwas ligated to the pSG-5 vector using T4 DNA ligase (New EnglandBiolabs, Beverly, Mass.) and transformed into DH5α competent cells(GIBCO, BRL Life Technologies, Gaithersburg, Md.) followingmanufacturer's instructions. Miniprep DNAs were analyzed and isolationof plasmid DNA performed. The nucleotide sequence of the light chaingene was determined using the dideoxy-nucleotide chain terminationmethod (Sanger et al., supra). The nucleotide sequence of the 5'untranslated region, mouse B72.3 light chain leader sequence, lightchain variable region, light chain constant region, and SV40 signalsequence was obtained. The nucleotide and amino acid residue sequencesare illustrated in FIGS. 25A and 25B and are given in the sequencelisting as SEQ ID NOs 152 and 153.

c. Transient Expression of b12 Heavy and Light Chain Genes in pSG-5Vectors in COS-7 Cells

1) Transient Expression of b12 IgG1 in COS-7 Cells

The human heavy and light chains in the separate pSG-5 expressionvectors were cotransformed and transiently expressed in COS-7 cells.COS-7 cells (SV40 transformed African Green Monkey Kidney Cells) providea rapid and convenient method to test the expression and function of theantibody genes. The COS-7 cells constituitively express the SV40 large Tantigen which supports the transient replication of episomes carryingthe SV40 origin of replication. The pSG-5 expression vector has an SV40origin of replication. Upon transfection into COS-7 cells, theexpression vectors are replicated in the nucleus to a high copy number,resulting in relatively high transient expression levels.

COS-7 cells were obtained from the American Type Culture Collection (CRL1651) and cultured in Dulbecco's modified Eagle's medium (DMEM),supplemented with 10% fetal bovine serum (GIBCO BRL, Gaithersburg, Md.)and 1% penicillin, and 1% streptomycin. Transfections were performedwith 10 μg of plasmid DNA per 100 mm tissue culture plate containing 1×10⁶ cells. The control plate was transfected with plasmid vector DNAwithout an insert. The plates were incubated at 37° C. aftertransfection. The supernatants were harvested at 48 hours and tested forgp120 binding specificity in an ELISA assay.

2) ELISA Assay for the Detection of Binding of b12 IgG1 to gp120

Supernatants from COS-7 transformants were tested for binding to gp120in an ELISA assay. Briefly, the ELISA plate was coated with recombinantIIIB gp120 antigen at a concentration of 1 μg/ml. The serially dilutedsupernatant containing the b12 antibody was added to the wells andincubated at 37° C. for 1 hour. After washing the plate to removeunbound antibody, a goat anti-human Ig Fc horse radish peroxidase (HRP)conjugated secondary antibody was added and incubated for an additionalhour. An OPD substrate for the HRP conjugated antibody was added and theHRP activity detected by determining the absorbance at 490 nm.

d. Insertion of the b12 Heavy Chain IgG1 into the pEE6 MammalianExpression Vector to Create pEe6HC BM 12

After confirmation that the antibody molecule expressed by the heavy andlight chain pSG-5 expression vectors bound gp120 as described in Example4c, the heavy chain was removed from the pSG-5 vector and ligated intothe pEE6 mammalian expression vector (Bebbington et al., Bio/Technology,10:169 (1992)). The pEE6 vector (Celltech, England) contains an HCMVpromoter and the glutamine synthetase gene (GS). The pEE6 vector waschosen because of the GS gene which serves as a selectable marker. CHOcells are devoid of GS activity and thus are dependent on a supply ofglutamine in the culture medium. Cells transfected with the pEE6 vectorcontaining the GS gene are able to synthesize glutamine from glutamateand can survive in the absence of glutamine in the culture medium. ForCHO cells, the addition of methyl sulfoxamine (MSX) leads toamplification of the transfected plasmid DNA.

The heavy chain pSG-5 vector was digested with EcoR I and Bgl II toremove the 5' untranslated region including the unique Kozak sequence,mouse heavy chain B72.3 leader sequence, and heavy chain variable andconstant regions from the pSG-5 vector. The pEE6 vector was alsodigested with EcoR I and BamH I. Both the vector and heavy chain DNAswere analyzed on a 0.7% low melting point agarose (LMPA) gel. The 3.5 kbheavy chain band and the 4.68 kb pEE6 vector band were excised from thegel and ligated together in the presence of the LMPA at 15° C. overnightwith 1 μl of T4 DNA ligase and 1 μl of 10× ligase buffer (New EnglandBiolabs, Beverly, Mass.). Upon ligation, the EcoR I site isreconstituted but the BamH I and BglII sites are destroyed. Prior totransformation, 5 μl of the ligated DNA in LMPA was diluted with 20 μlof TCM buffer (10 mM tris, 10 mM CaCl₂, and 10 mMMgCl₂). Only 10 μl ofthe 25 μl was used for the transformation. The ligated circular plasmidDNA construct was transformed into maximum efficiency DH5α competentcells. The standard protocol for transformation was used, wherein theDNA and 100 μl of the competent bacterial mix (GIBCO BRL, Gaithersburg,Mass.) were incubated on ice for 20 minutes and heat shocked at 42° C.followed by incubation on ice for 2 minutes. About 900 μl of SOC (GIBCOBRL, Gaithersburg, Mass.) was added to the transformation. Only 100 μlof the 1000 μl of the transformed cells was plated on LB withcarbenicillin plates (carbenicillin at 50 μg/ml). The plates wereincubated at 37° C. overnight. Twelve individual colonies were pickedfor miniprep analysis. Several diagnostic digests confirmed the presenceof the heavy chain insert. Plasmid DNA was isolated on a CsCl gradient(Sambrook et al., supra). The nucleotide and amino acid residuesequences are illustrated in FIGS. 27A through 27E and the nucleotideand amino acid residue sequences are given in the sequence listing asSEQ ID NOs 154 and 155.

e. Insertion of the b12 Light Chain into the pEE12Mammalian ExpressionVector

The light chain was ligated into the pEE12 vector (Celltech, England)from the pSG-5 vector involving similar steps as described in Example 4dfor the heavy chain. The pEE12 vector has a human CMV promoter forexpression of the light chain, a polylinker to provide cloning sites,and a polyadenylation signal for termination of transcription. Thevector also contains the GS selectable marker gene, whose expression iscontrolled by an SV40 early promoter at the 5' end of the GS gene, anintron, and a polyadenylation signal at the 3' end of the GS gene.

1) Preparation of Modified b12 Light Chain

The 5' PCR primer was designed to replace the EcoR I cloning site with aHindIII cloning site. The 3' PCR primer maintained the EcoR I cloningsite.

The primer pair, LC-5 (SEQ ID NO 167) and LC-2 (SEQ ID NO 165,), wasused in the PCR reaction as described in Example 4al to amplify the Fabb12 light chain gene and incorporate HindIII and EcoR I cloning sitesinto 5' and 3' ends of the fragment, respectively. The b12 pSG-5 vectorcontaining the b12 light chain was used as the template in the PCRreaction. For the PCR reaction, 1 μl containing 100 ng of b12 pSG-5 DNAwas admixed with 10 μl of 10× PCR buffer in a 0.5 ml microfuge tube. Tothe DNA admixture, 8 μl of a 2.5 mM solution of dNTPs (dATP, dCTP, dGTP,dTTP) was admixed to result in a final concentration of 200 micromolar(μM) of each dNTP. 1 μl (equivalent to 20 pM) of the LC-5 primer and 1μl (20 pM) of the 3' backward LC-2 primer was admixed into the DNAsolution. To the admixture, 73 μl of sterile water and 2.5 units of TaqDNA polymerase was added. Two drops of mineral oil were placed on top ofthe admixture and 35 rounds of PCR amplification in a thermocycler wereperformed. The amplification cycle consisted of 52° C. for 1 minute, 72°C. for 2 minutes and 94° C. for 0.5 minutes.

The resultant PCR modified b12 light chain DNA fragment was purified byelectrophoresis in a 2.5% Nu-Sieve agarose gel (FMC). The area in theagarose containing the modified b12 light chain DNA fragment wasisolated from the agarose.

2) Insertion of the Modified b12 Light Chain into the pEE12 MammalianExpression Vector

The modified b12 light chain purified PCR product and the pEE12 vectorwere digested with HindIII and EcoR I in separate reactions. Thedigested DNAs were analyzed on an LMPA gel, the DNA excised, and ligatedtogether in the presence of the LMPA gel as described for the heavychain construct in Example 4d. The ligation products were transformedinto DH5α competent cells, minipreps analyzed, and DNA prepared asdescribed for the heavy chain constructs in Example 4d.

f. Insertion of the Modified b12 Heavy Chain into the pEE12 MammalianExpression Vector Containing the b12 Light Chain to Create theCombinatorial Vector pEe12 Combo BM 12

A heavy chain cassette comprising the HCMV promoter, enhancer elements,heavy chain gene, and polyadenylation signal were removed from the pEE6vector and inserted into the pEE12 vector containing the b12 light chaingene, prepared in Example 4e, to generate the combinatorial construct,peel2 Combo BM 12, containing both the b12 light and heavy chain genes(FIG. 28).

The heavy chain cassette was removed from the pEE6 vector by digestionwith BglII and Sal I. The pEE12 vector containing the light chain gene,prepared in Example 4e, was also digested with BglII and Sal I. Theheavy chain cassette and the pEE12 vector containing the light chaingene from Example 4e were ligated together at the BglII and Sal I sitesas described in Example 4d. The combinatorial construct was transformedinto DH5α competent cells and miniprep DNA was analyzed for the presenceof the heavy and light chains as in Example 4d. The nucleotide sequenceof the heavy and light chain genes was determined. The nucleotidesequence of peel2 Combo BM 12, the pEE12 vector containing the b12 heavyand light chain genes is given in the sequence listing as SEQ ID NO 156and is illustrated in FIGS. 29A through 29R.

g. gp120 Binding of b12 IgG1 Antibody Expressed from the Heavy and LightChain Genes in the Combinatorial Vector pEe12 Combo BM 12

The combinatorial pEe12 Combo BM 12 vector containing both the heavy andlight chain genes was used to transfect CHO cells. Stable clones wereselected in Glasgow Minimal Essential Media (GIBCO) supplemented with10% dialyzed fetal bovine serum and 50 μM methyl sulfoxamine (MSX).Several clones were isolated and expanded in 6-well cluster dishes. Thesupernatants of subconfluent cultures were harvested and tested by ELISAfor binding to gp120 as described in Example 4c2. The clone producingthe highest levels of b12 IgG1 as determined by ELISA with gp120 IIIBwas chosen for further study. The antibody was purified by affinitychromatography using protein A as described in Sambrook, et al., supra.The affinity of b12 IgG1 for gp120 IIIB as measured by surface plasmonresonance as described in Example 2b6c is 1.3×10⁹ M⁻¹.

5. Neutralizing Activity of Recombinant b12 Whole IgG1 Antibody (b12IgG1) Against HIV-1 In Vitro

The key issue in producing antibodies to HIV-1 for therapeutic orprophylactic purposes is that they should be highly potent (of highaffinity and neutralizing ability) and be cross reactive with a widerange of primary clinical (field) isolates. These are generally twoopposing characteristics. The ability of b12 whole IgG1 antibody (b12IgG1) to neutralize the infectivity of laboratory strains of HIV-1 and awide variety of primary clinical isolates has been examined in p24 ELISAassays, microplaque assays, and by syncytial formation assays.

The primary clinical isolates used as a source of HIV-1 virus in theseassays came from various regions of the world by three organizations:the World Health Organization (WHO), the Henry M. Jackson Foundation forthe Advancement of Military Medicine (HMJFAMM), and the NationalInstitute of Allergy and Infectious Diseases (NIAID). Isolates from theWHO Network for HIV-1 Isolation and Characterization were obtainedthrough the AIDS Research and Reference reagent Program, Division ofAIDS, NIAID, NIH. Isolates from HMJFAMM were provided by Dr. JohnMascola, Walter Reed Army Institute of Research, Rockville, Md. and Dr.Francine McCutchan, Henry M. Jackson Research Laboratory, Rockville Md.Isolates from NIAID were kindly provided by Dr. Jim Bradac, Division ofAIDS, NIAID, NIH.

The EIV-1 viruses were collected from various regions of the world,expanded in mitogen-stimulated peripheral blood mononuclear cells (PBMC)(Mascola et al., J. Infect. Dis., 169:48-54 (1994)), and culturesupernatants containing infectious virus were stored in centralrepositories at -70° C. The designation of viruses into clades was madeon the basis of sequence information based on the gag gene or on theV2-C5 region of gp120, or in some cases, after heteroduplex mobilityanalysis (Louwagie et al., AIDS, 7:769-772 (1993) and Delwart et al.,Science, 262:1257-1261 (1993)).

The HIV-1 viruses include a set of 14 primary isolates which contain ahigh proportion of isolates which are relatively refractory to antibodyneutralization by sera from other HIV-1 infected individuals (Wrin etal., J. Acq. Imm. Def. Synd., 7:211-219 (1994)), 12 primary infantisolates obtained at birth or within two weeks of age, and 69international isolates belonging to 6 different clades.

Several different neutralization assays were performed because HIV-1neutralization by antibody shows considerable variation depending uponthe assay used and the precise experimental conditions such as inoculumsize and incubation time of virus and antibody (D'Souza et al., AIDS,8:169-173 (1994)). By performing neutralization assays on a range oflaboratory and primary isolates in a number of different laboratories,it has been demonstrated that b12 IgG1 is a highly potent neutralizingantibody effective against a wide breadth of isolates.

a. Quantitative Neutralization of HIV-1MN and IIIb by b12 IgG1 asMeasured in a Plaque Assay

b12 IgG1 was initially tested for its ability to neutralize the HIV-1laboratory strains MN and IIIB in a plaque formation assay inlaboratories which recently tested a panel of monoclonal antibodies aspart of the NIAID/WHO Antibody Serological Project (D'Souza et al.,supra).

b12 IgG1 showed 50% neutralization titers of 3 ng/ml for the MN strainand 7 ng/ml for the IIIB strain using plaque formation (Hanson, et al.,J. Clin. Microbiol., 28:2030-2034 (1990)) to determine the ability ofthe antibody to inhibit infectivity of the HIV-1 strains.

b. Quantitative Neutralization of HIV-1MN and IIIb by b12 IgG1 asMeasured by Syncytial Formation

b12 IgG1 showed 50% neutralization titers of 20 ng/ml for both MN andIIIB strains using syncytial formation as the reporter assay asdescribed in Example 3b (Nara et al., AIDS Res. Human Retroviruses,3:283-302 (1987)).

The syncytial formation assay was performed as described in Example 5c.Briefly, virus was grown in H9 cells. For infectivity measurement,monolayers of CEM-SS target cells were cultured with 100-200 syncytialforming units (SFUs) of virus, in the presence or absence of antibody,and the number of syncytia determined after 3-5 days of incubation. Theassays were repeatable over a virus-surviving fraction range of 1 to0.001 within a 2 to 4-fold difference in the concentration of antibody(P<0.001).

c. Neutralization of Primary Virus Isolates by b12 IgG1 as Measured bythe p24 ELISA Assay

The ability of b12 IgG1 to neutralize infectivity of PBMCs by HIV-1virus was quantitatively measured in the p24 ELISA assay (Daar et al.,Proc. Natl. Acad. Sci. U.S.A., 87:6574-6578 (1990) and Ho et al., J.Virol., 65:489-493 (1991)). The p24 ELISA assay is further described inExample 3a.

1) Neutralization of Ten Primary Virus Isolates by b12 IgG1

HIV-1 viruses were isolated from 10 individuals from various locationsin the U.S. and with varying disease status. The HIV-1 viruses had beencultured only once or twice in peripheral blood mononuclear cells(PBMCs). Vital stocks were grown in PBMCs and the assay was performed inPBMCs.

Briefly, HIV-1 virus at 50 TCID₅₀ and varying concentrations of b12 IgG1were incubated together for 30 min at 37° C. before addition toPHA-stimulated PBMCs. HIV-1 virus replication was assessed afterincubation for 5 to 7 days by p24 ELISA measurement as described inExample 3a. HIV-1 virus positive controls used in this assay were themolecularly cloned HIV-1 virus JR-CSF and the HIV-1 isolate JR-FL(O'Brien et al., J. Virol., 66:3125-3130 (1992), O'Brien et al., Nature,348:69-73 (1990), and O'Brien et al., J. Virol., in press (1994)).Stocks of JR-CSF were prepared by infection of PBMC with supernatantsinitially obtained by DNA transfection. HIV-1 IIIB and HIV-1MN areviruses with an extensive history of passage in transformed T-cell lines(Robert-Guroff et al., Nature, 316:72-74 (1985)). Stocks of thesestrains grown in H9 cells were passaged in mitogen-stimulated PBMC toprepare viruses that had been grown in the same cells as the primaryviruses, to eliminate the influence of any host cell-dependent epigenicfactors on virus neutralization (Wrin, et al., J. Acq. Imm. Def. Synd.,7:211-219 (1994)). The stock of PBMC-grown MN was a gift from A. N.Conley (Merck Research Labs).

2) Neutralization of 12 Primary Infant Isolates by b12 IgG1

b12 IgG1 was also tested for the ability to neutralize infectivity of apanel of 12 primary infant isolates in the p24 ELISA assay. Virusisolates were obtained from 12 infants born to HIV-1 seropositivemothers; 7 were obtained at birth and 5 between birth and 14 days ofage. All the infants were from California. Virus was isolated frompatient PBMCs by coculture with PBMCs from healthy seronegative donors.Viral stocks were prepared by passaging the last positive culturedilution once into PBMCs. All of the isolates, except one (isolate 7),were non-syncytial inducing in MT2 cells and therefore could not beassayed in the syncytial forming assay as herein described. HIV-1 virusfrom these stocks was grown in PBMCs and neutralization assessed usingPHA-stimulated PBMCs as indicator cells and determination ofextracellular p24 as the reporter assay essentially as described inExample 3a (AIDS Clinical Trials Group Virology manual for HIVLaboratories, Department of AIDS Research, NIAID, NIH, version 2.0(1993)).

Serial dilutions of b12 IgG1 (0.3 to 20 μg/ml) were incubated with 20TCID₅₀ or 100 TCID₅₀ virus in triplicate for 2 hours at 37° C. beforeaddition to PHA-stimulated PBMCs. Virus replication was assessed after 5days by p24 ELISA measurement. Neutralization was expressed as either a50% or 90% reduction in p24 antigen as compared to values observed inthe absence of antibody (Table 6).

d. Neutralization of Primary Virus isolates by b12 IgG1 as Measured in aMicroplaque Assay

A quantitative microplaque assay to measure the reduction of infectivityof primary clinical isolates of HIV-1 in the presence of the b12 IgG1and pooled human plasma was performed as described in Hanson et al., J.of Clin. Microb., 2030-2034 (1990). The set of primary clinical isolateswas chosen to contain a high proportion of isolates which are relativelyrefractory to antibody neutralization by sera from other HIV-1 infectedindividuals (Wrin et al., J. Acq. Imm. Def. Synd., 7:211-219 (1994)).Viruses were grown in PBMCs and the assay carried out in MT2 cells. Thislimits study to viruses which grow in this cell line but provides anadditional measure of neutralization.

Primary clinical isolates of HIV-1 were isolated from frozen peripheralblood lymphocytes obtained from seropositive donors as described inGallo et al., J. of Clin. Microb., 1291-1294 (1987) and cultivated inperipheral blood mononuclear cells (PBMC). Briefly, HIV isolates wereobtained by incubating frozen HIV-infected patient PBMCs withseronegative donor PBMCs in RPMI-1640 medium containing 20%heat-inactivated fetal bovine serum, 2 μg/ml polybrene, 5%interleukin-2, and 0.1% anti-human leukocyte interferon. The cultureswere fed with fresh donor PBMCs once a week, and the supernatants wereassayed for the presence of reverse transcriptase (RT) activitybeginning at day 11. The cultures were considered positive if, for 2consecutive weeks, the RT counts were >10-fold higher than those in thecultures of the seronegative donor PBMCs alone.

The resultant RT-positive virus isolates were tested for cytolysis inthe MT4 (α-4 clone) (Hanson et al., supra). Cytolysis in MT4 is arequirement for viruses to be usable in the subsequent MT2 microplaqueassay system. Supernatant fluids from the primary PBMC isolationcultures were used to infect expanded cultures of phytohemagglutinin(PHA)-stimulated PBMCs from healthy seronegative blood donors. Theseinfected PBMC cultures were grown in RPMI-1640 medium supplemented with15% fetal bovine serum, 5% interleukin-2, 0.1% anti-α interferon, 2μg/ml polybrene, 50 μg/ml gentamicin, 100 U/ml penicillin, and 100 μg/mlstreptomycin. The crude supernatants were harvested after 7 days andfrozen as viral stocks at -70° C.

The primary clinical isolates of HIV-1 used in this microplaque assayare given in Table 6. VL134, VL648, and VL025 are viruses isolated frominfected mothers in New York in 1992; UG266 and UG274 are clade Disolates which were a gift from John Mascola the Division ofRetrovirology, Walter Reed Army Institute of Research; the remainingviruses were isolated from homosexual males in California in 1992. Thepooled human plasma preparation, containing neutralizing antibody, wasderived from 13 HIV-1 positive individuals selected for highneutralization titer against the MN isolate. The laboratory HIV-1strains MN and IIIb were propagated in H9 cells as controls in themicroplaque assay.

b12 IgG1 and a pool of human plasma from 13 HIV-1 seropositive patientswere used as the source of neutralizing antibodies in a 96-wellmicrotiter plaque reduction assay as described by Hanson et al., supra.Briefly, 3-fold serial dilutions of the b12 IgG1 or heat-inactivatedpooled patients' plasma were combined in quadruplicate with an equalvolume containing 20 plaque-forming units (PFU) of HIV-1 virus per welland incubated for 18 hours at 37° C. Negative control wells alsocontained 50% normal human serum pool with no patient immune serum.After the 18 hour incubation of Fabs or serum and virus, 90,000 MT2cells were added per well and incubated at 37° C. for 1 hour. SeaPlaqueAgarose in assay medium at 39.5° C. was then added to a finalconcentration of 0.8%. While the warm agarose was still molten, themicrotiter plates were centrifuged at 20° C. for 20 minutes at 500 × gto form cell monolayers. The plates were incubated for 6 days at 37° C.and then stained 18 to 24 hours with 50 μg/ml propidium iodide. Thefluorescent plaques were counted with transillumination by a 304 nmultraviolet light source using a low-power stereo zoom microscope.Inhibition of infectivity, or neutralization titer, is defined as theμg/ml of Fab or the plasma dilution giving 50% inhibition of plaquecount as compared with controls without antibody. This dilution wasinterpolated between data points.

e. Results of the Neutralization Assays by b12 IgG1 with LaboratoryVirus Isolates

Results of the ability of the b12 IgG1 to neutralize laboratory virusisolates in both the plaque and syncytial formation assays suggest theantibody is approximately two orders of magnitude more potent than otherCD4 site antibodies in the WHO/NIAID Project and comparable to the bestantibodies directed to the V3 loop of gp120. However, whereas antibodiesdirected to the V3 loop of gp120 are strongly strain specific, b12 IgG1is roughly equally effective against MN and IIIB. The b12 IgG1 antibodyis comparable in potency to a CD4-IgG molecule in these assays (Example3c). In a separate assay using p24 production to determine infectivity(Daar et al., Proc. Natl. Acad. Sci. U.S.A., 87:6574-6580 (1990) and Hoet al., J. Virol., 65:489-493 (1991)), 50% neutralization titers of lessthan 40 ng/ml were found for both the MN and IIIB laboratory strains.

f. Results of the Neutralization Assays by b12 IgG1 with Primary VirusIsolates

b12 IgG1 showed essentially complete neutralization of 7 of 10 isolatesat 5 μg/ml with all the isolates showing 50% neutralization at ≦1 μg/mlas determined in the p24 reporter assay (FIG. 21).

The inhibition of infectivity, or neutralization titer, for b12 IgG1 andthe pooled HIV seropositive human plasma from 13 donors is given inTable 6. The neutralization titer for each of the viral isolates isexpressed as the minimum μg/ml of b12 IgG1 required for 50% inhibitionof plaque count as compared to the controls. The neutralization titerfor each of the viral isolates is expressed as the minimum titer of thepooled HIV seropositive human plasma from 13 donors required for 50%inhibition of plaque count as compared to the controls.

                  TABLE 6    ______________________________________                                   pooled human                        b12 IgG1 50%                                   plasma: dilu-            host        neutralization                                   tion for 50%;    virus   cell        titer (μg/ml)                                   neutralization    ______________________________________    IIIB    H9          0.007       1:767    MN      H9          0.003         1:24,000    VL135   PBMC        10         1:44    UG274   PBMC        0.7        1:37    VL134   PBMC        5.6        1:30    VL596   PBMC        8.5        1:17    UG266   PBMC        3.8        1:12    VL434   PBMC        22         1:10    VL172   PBMC        >200       1:10    VL750   PBMC        >200       1:10    VL069   PBMC        >50        <1:10    VL077   PBMC        >200       <1:10    VL114   PBMC        <7.4       <1:10    VL263   PBMC        5.0        <1:10    VL648   PBMC        16.7       <1:10    VL025   PBMC        16.7       <1:10    ______________________________________

The b12 IgG1 was able to neutralize ten of the fourteen primary clinicalisolates assayed at concentrations of ≦50 μg/ml as measured as the μg/mlrequired for 50% inhibition of plaque count as compared to the controls(Table 6). Pooled human plasma was able to neutralize 5 of the 14primary clinical isolates assayed at >1:10 dilution as measured as thedilution required for 50% inhibition of plaque count as compared to thecontrols without antibody.

Table 6 shows that four isolates, which were not neutralized even by a1:10 dilution of pooled human plasma, were neutralized by b12 IgG1.Mostof the viruses reported in Table 6 were isolated from U.S. donorsalthough two, both of which are neutralized by b12 IgG1, were fromUgandan donors and assigned to clade D.

Results of neutralization of 12 infant primary isolates with b12 IgG1 asdetermined by p24 ELISA measurements are given in Table 7.

                  TABLE 7    ______________________________________               b12 IgG1               Antibody Concentration (μg/ml)    Infant Isolate                 50% inhibition                            >90% inhibition    ______________________________________    1            20         >20    2            1.25       >20    3            <0.3       0.3    4            <0.3       0.6    5            2.5        20    6            5          >20    7            5          >20    8            <0.3       0.3    9            0.3        5    10           0.3        2.5    11           <0.3       0.6    12           <0.3       0.3    ______________________________________

As shown in Table 7, b12 IgG1 achieved 90% neutralization for 8 of 12infant isolates at concentrations of ≦20 μg/ml in the p24-based assay.All 12 isolates were 50% neutralized in the range of 0.3 to 20 μg/mlwith the majority being neutralized at <5 μg/ml. In contrast, a pooledhyperimmune globulin product HIVIG achieved 90% neutralization of only 3or 12 isolates within a concentration range up to 100 μg/ml. HIVIG is ahyperimmune IgG preparation obtained from the pooled plasma of selectedHIV-1 asymptomatic seropositive donors meeting the following criteria:presence of p24 serum antibody titers >128, CD4 lymphocyte count ≧400cells/μl and the absence of p24 and hepatitis B surface antigen byenzyme immunoassay (Cummins et al., Blood, 77:1111-1114 (1991)). TheHIVIG used in these experiments was lot number IHV-50-101 (NorthAmerican Biologicals).

HIV-1 neutralization by antibody shows considerable variation dependingupon the assay used and precise experimental conditions such as inoculumsize and incubation time of virus and antibody (D'Souza et al., supra).However, by carrying out neutralization on a range of laboratory andprimary isolates in a number of assays in different laboratories, wehave shown that b12 IgG1 is a highly potent neutralizing antibodyeffective against a wide breadth of primary isolates. The resultsclearly demonstrate that, although primary isolates may be moredifficult to neutralize by antibody than laboratory strains, they arenot intrinsically resistant (Conley et al., Proc. Natl. Acad. Sci.U.S.A., 91:3348-3353 (1994)). The potency of b12 IgG1 against themajority of U.S. isolates is in a concentration range (≦5 μg/ml) whichcould be achieved in vivo in passive immunotherapy. Furthermore, theaffinities of recombinant antibodies displayed on phage can be enhancedby mutagenesis and selection in vitro and this strategy has been used toconsiderably improve the potency and breadth of reactivity of Fab b12(Barbas et al., Proc. Natl. Acad. Sci., U.S.A., 91:3809-3812 (1994)).For optimal potency and strain cross-reactivity for passiveimmunization, a cocktail of in vitro improved antibodies may be mostappropriate.

The results have implications for passive immunization and vaccinedesign. The ability of b12 IgG1 to neutralize a range of primaryisolates implies conservation of a structural feature associated withthe CD4 binding site of gp120 which is accessible to antibody andimportant for neutralization. A vaccine might seek to present thisfeature to the immune system. Clearly, the feature is present onrecombinant gp120 since b12 was affinity selected from a library usingthis molecule. However, b12 and related antibodies formed only a smallpart of the repertoire affinity selected from this library byrecombinant gp120.Most of the antibodies obtained were far less potentin neutralization even though they were also directed to the CD4 bindingsite, were cross-competitive with b12 for binding to recombinant gp120and had similar affinities to b12 (Barbas et al., Proc. Natl. Acad.Sci., U.S.A., 89:9339-9343 (1992), Barbas et al., J. Mol. Biol.,230:812-823 (1993), and Example ). Therefore, recombinant gp120 appearsto present the b12 epitope in conjunction with several other weaklyneutralizing and overlapping epitopes and its efficacy as a vaccine maysuffer. Interestingly, evidence from antibody binding to infected cellssuggests that b12 does recognize a native conformation of gp120 moreeffectively than other CD4 binding site antibodies (Example 7). In anycase, b12 IgG1 and the library approach could be useful in vaccine andpassive immunization evaluation. The ability of a candidate vaccine topreferentially bind b12 and/or preferentially select potent neutralizingantibodies from libraries should be positive indicators for vaccinedevelopment.

5. Determination of the Relationship Between the Epitopes Recognized byFabs with Purified HIV-1 Antigens

The Fabs show a spectrum of neutralizing abilities as described inExample 5. It was therefore sought to determine if the epitopesrecognized by individual Fabs could be distinguished from each other,and if possible, determine how the epitopes recognized by the individualFabs related to neutralization.

a. Competitive ELISA between Fabs and b13 Whole IgG1 Antibody forBinding to gp120

The first method to distinguish between the epitopes bound by the Fabsof this invention was to compare the epitope recognized by the Fab b13with the other Fabs. The Fab b13 had been spliced to the Fc region ofIgG1 to generate a whole IgG1 molecule and therefore contains the Fcregion of the IgG1 antibody. The other Fabs do not contain the Fc regionof the IgG1 antibody. The binding of the b13 IgG1 could therefore bedistinguished from the binding of other Fabs by using a labeled anti-Fcreagent in competition ELISA. A competition ELISA in which the Fabs b3,b6, b11, b12, and b14 competed with b13 IgG1 for binding to immobilizedgp120 was performed.

Competitive ELISAs were performed between the Fabs b3, b6, b11, b12, andb14 and the b13 whole IgG1 antibody. The whole antibody was obtained bysplicing constant domain genes with the b13 Fab and expressing theprotein in Chinese Hamster Ovary cells (CHO) as described in Example 4(Bender et al., supra and in Example for the Fab b12). The ELISA wasperformed as described above in Example 2b6b. Briefly, microtiter wellswere coated with 0.1 μg/ml of gp120 derived from the HIV-1 strain LAI in0.1M bicarbonate buffer at pH 8.6. Soluble or free Fab fragments wereserially diluted from 1:100 to 1:32,000 in 0.5% BSA/0.025% Tween 20/PBS.The dilution of b13 IgG1 was held constant at 1:10,000 in 0.5%BSA/0.025% Tween 20/PBS. The b13 IgG1 and Fabs were admixed, added tothe gp120-coated microtiter wells and maintained for 120 minutes at 37°C. After maintenance, the wells were carefully washed ten times with0.05% Tween 20/PBS. The amount of b13 IgG1 antibody bound to the plateafter washing was detected using a peroxidase-labeled antibody specificfor the Fc portion of IgG1 contained on the b13 antibody.

Results of this assay indicated that the Fabs b3, b6, b11, b12, and b14are competitive with b13 IgG1 for binding to gp120 indicating that theepitopes recognized by the individual Fabs are probably either proximalor identical to the epitope recognized by the b13 IgG1. A controlanti-tetanus toxoid Fab did not compete with IgG1 b13 in this assay.

Competition monitored in an ELISA format showed that all of the Fabscompete with the b13 Fab as a whole IgG. There is also an indicationthat Fabs b12 and b13 are distinct in that they are somewhat lesseffective in cross-competition than the other members of the panel.

b. Epitome Similarity Determination Between the Fabs in Binding to gp120Using BIAcore

A more precise method for determining the similarity of epitopes wasperformed using the BIAcore. The procedure adopted here was toimmobilize a polyclonal anti-human F(ab')₂ on the sensor chip and usethis to capture the individual Fabs. An Fab of this invention wasinjected and captured by the polyclonal anti-human F(ab')₂. The capturedFab was then used to bind gp120 derived from the HIV-1 strain LAI. Thecaptured Fab would thus bind the gp120 at its respective epitope. Asecond Fab of this invention was then injected. A response in theBIAcore assay after injection of the second Fab indicates that bindinghas occurred. If the second Fab injected recognizes the same or similarepitope on the gp120 as the first Fab, no response would occur. Noresponse would therefore indicate that the two Fabs tested in the assaycompeted for binding to the same or similar epitope on gp120.Alternatively, a response in the assay suggests that the epitopesrecognized by the two Fabs are distinct from one another and thatbinding of the second Fab to gp120 to a second epitope is possible inthe presence of the first Fab. A response would therefore indicate thatthe two Fabs tested in the assay did not compete for binding to the sameor similar epitope.

The precise epitope similarity determination with the BIAcore wasperformed as follows. A flow rate of 5 μl/min of PBS, pH 7.4 wasestablished and the biosensor chip was activated by injecting 30 μl ofactivation solution (Pharmacia Biosensor, 50% 0.2MN-ethyl-N'-(e-diethylaminopropyl)-carbodiimide, 50%N-hydroxysuccinimide). The flow rate was then adjusted to 10 μl/min andthe antigen was injected in 10 mM sodium acetate buffer, pH 4.5. Fortyμl of goat anti-human F(ab')₂ (Pierce) at a concentration of 40 μg/ml in10 mM sodium acetate buffer, pH 4.5 was injected to give a finalimmobilization of 10000 Response Units (RU). The chip was then blockedfrom any further immobilization by injecting 30 μl of 1M ethanolamine,pH 8.5 (Pharmacia Biosensor). The flow rate was adjusted to 1 μl/min and4 μl of the first Fab at a concentration of 100 μg/ml was injected,immediately followed by 4 μl of an anti-cytomegalovirus Fab at aconcentration of 150 μg/ml to block any remaining binding sites on theimmobilized goat anti-human F(ab')₂. Next, 4 μl of gp120 at aconcentration of 10 μg/ml was injected followed by 4 μl of the secondFab at 100 μg/ml. The assay was performed with a combination of all ofthe Fabs to give a mosaic of binding patterns. The entire surface wasregenerated with 25 μl of 60 mM HCl so that the next cycle could beperformed.

Table 8b indicates the results of the epitope similarity determinationby BIAcore. Table 8a shows the positive and negative controls for theclones used. The positive controls are the RU levels obtained when thefirst Fab used is the clone indicated and the second Fab is ananti-gp120 V3-loop Fab. The Fabs of this invention compete with solubleCD4 for binding to gp120. The second Fab, an anti-gp120 V3-loop Fab,neither competes with soluble CD4 nor competes with anti-CD4 site Fabsand therefore would react with a different epitope than the Fabs of thisinvention. As can be seen from the table, all positive controls resultin significant values of 125 or more, indicating the validity of thetechnique to distinguish between non-identical epitopes. The negativecontrols are the values obtained when the same Fab is injected twice.This gives the background values for each Fab. These values weresubtracted from all subsequent experiments in order to give true values.

An epitope map, Table 8b, was then constructed. ND indicates that thiscombination of Fabs was not performed. It can be seen from this map thatFabs b3, b6, b11, and b14 form a set which compete highly effectivelywith one another for binding to a similar or the same epitope. For themost part, a member of the set competes for binding as well with anothermember as it does with itself (RU=0). On the other hand, b12 and b13appear somewhat different in that while they compete for binding withmembers of the above set, they do not compete as effectively as theother Fabs within the set. Further, competition for binding to the sameor similar epitope between b12 and b13 is incomplete. This suggests thatthe epitopes of Fabs b12 and b13 are sufficiently dissimilar from thoseof the other four and from each other, to allow detectable binding whenthey are used in combination with any of the other Fabs. It maytherefore be concluded that clones b3, b6, b11, and b14 bind the same orsimilar epitopes, with Fabs b12 and b13 bind to epitopes which can bedistinguished from the other epitopes in this assay.

                  TABLE 8a    ______________________________________    Fab        b3     b6      b11   b12  b13   b14    ______________________________________    POSITIVE   129    128     131   125  135   134    CONTROL (RU)    NEGATIVE   24     38      ND    17   15    ND    CONTROL (RU)    ______________________________________     ND indicates that this combination of Fabs was not performed.

                  TABLE 8b    ______________________________________             Fab 1    Fab 2      b13    b12          b6  b3    ______________________________________    b14        30     24           14  0    b11        54     28           14  0    b3         26     29            0  0    b6         21     17            0  ND    b12        22     0            ND  ND    ______________________________________     ND indicates that this combination of Fabs was not performed.

c. Comparison of Fab Epitopes with Wild-type and Mutant Forms of gp120Using ELISA with gp120 in the Solid Phase

Epitope similarity determinations of the panel of Fabs was performedwith a panel of HXBc2 gp120 mutants of the HIV-1 strain LAI. Conservedresidues of gp120 were altered to generate the HXBc2 gp120 mutants. Theinteraction between the mutants and Fabs was investigated to examinebinding specificity differences between the Fabs at greater resolution.The HXBc2 gp120 mutants used in this assay had been previouslycharacterized with respect to gp160 precursor processing, gp120-gp41association, and CD4 binding ability (Olshevsky et al., J. Virol.,64:5701-5707 (1990)). Both wild type and mutant gp120s were tested fortheir ability to bind a saturating concentration of each Fab.

The epitope determination with wild-type and mutant gp120 was performedwith HIV-1 envelope glycoproteins from culture supernatants of COS-1cells transfected with plasmids expressing either wild-type or mutantgp120 from the HXBc2 clone. Microtiter wells were coated with theantibody D7324 (Aalto BioReagents; Dublin, Ireland) which binds to theconserved 15 amino acid sequence at the carboxy terminus of gp120. Thewild-type or mutant gp120 were thus captured onto the surface ofmicrotiter wells by binding to the D7324 antibody. A reference HIV-1positive human serum pool at a 1:3000 dilution in 0.5% Tween 20 wasassayed for binding to the wild-type and mutant gp120s by incubating theserum pool with the immobilized gp120. The bound antibody was detectedby a second enzyme conjugated antibody. The reading obtained with theHIV-1 positive human serum pool, N=4, was used as the reference valuefor each mutant. The Fabs of this invention were then assessed forbinding to the wild-type and mutant gp120s and the ratio of the Fab toreference serum was determined for each gp120 mutant (Table 9). Theaverage ratio for the entire panel of Fabs was calculated and anyindividual ratio deviating from the mean by less than 0.5 times wasconsidered to indicate a gp120 amino acid change that decreased Fabrecognition, while those deviating by more than 2.0 times indicated anamino acid change that enhanced Fab recognition. In this way, a map ofmutations affecting the binding of the Fab to gp120 was obtained foreach clone essentially as previously described (Helseth et al., J.Virol., 65:2119-2123 (1991) and Olshevsky et al., supra).

                  TABLE 9    ______________________________________           Fab    Mutation B3      B6      B11   B12   B13   B14    ______________________________________    45 W/S   1.60    0.61    0.50  0.68  1.20  0.28    113 D/A  1.46    1.73    1.89  1.13  0.99  0.00    113 D/R  1.40    1.50    1.61  0.67  0.71  0.00    NO V1/V2 1.07    1.48    1.42  0.23  0.86  1.68    NO V1/V2/V3             2.05    1.48    1.94  0.47  0.95  1.60    NO V3    1.88    1.64    1.92  0.46  1.08  1.72    183/184 PI/SG             0.82    0.73    0.69  0.33  0.92  0.32    207 K/W  1.15    1.57    1.19  2.54  1.30  1.36    252 R/W  1.58    1.52    1.58  1.65  1.39  2.04    256 S/Y  0.64    0.14    0.33  0.82  1.15  0.00    257 T/R  0.08    0.59    0.00  0.76  0.22  0.00    257 T/A  0.86    0.93    0.75  0.99  0.68  0.40    257 T/G  0.91    0.70    1.14  0.74  0.75  0.00    262 N/T  1.06    0.64    1.19  0.62  0.72  0.24    269 E/L  0.73    0.48    0.45  0.78  0.83  0.20    314 G/W  0.59    0.36    0.39  0.65  0.71  0.28    356 N/I  0.67    0.66    0.39  0.92  0.80  0.52    368 D/R  0.19    0.18    0.00  0.04  0.00  0.00    368 D/T  0.28    0.20    0.00  0.03  0.02  0.00    370 E/R  0.01    0.25    0.17  0.07  0.00  0.00    370 E/Q  0.25    0.89    0.58  0.46  0.14  0.00    384 Y/E  1.21    1.02    1.11  0.25  0.02  0.88    386 N/Q  0.88    0.59    0.31  1.05  0.01  0.36    395 W/S  0.92    0.59    0.47  1.00  1.05  0.12    427 W/S  1.57    1.11    1.53  0.63  0.98  0.00    435 Y/S  1.93    1.16    1.58  1.41  1.24  2.04    450 T/N  0.62    0.48    0.58  0.75  0.75  0.60    457 D/A  0.62    0.39    0.44  0.28  0.62  0.20    457 D/R  0.84    0.55    0.92  0.32  0.58  0.56    470 P/L  0.80    0.64    0.72  0.72  0.18  0.24    475 M/S  0.06    1.02    0.33  1.50  1.39  0.92    477 D/V  0.50    0.09    0.00  0.07  0.52  0.00    ______________________________________

The general patterns observed are broadly similar to many CD4 siteantibodies and of soluble CD4. Fab b12 is distinguished by its decreasedbinding to a mutant in which the V1 and V2 loops are deleted. This mayor may not be related to the enhanced neutralizing ability of Fab b12.However, it is clear that the V1 and V2 loops and the V3 loop can affectantibody binding to the CD4 binding site either by direct contact or bytransmitted conformational effects.

Sensitivity to certain mutations in residues, particularly towards theC-terminus of gp120, has previously been associated with CD4 bindingsite antibodies (Thali et al., J. Virol., 66:5636-5641 (1992) and. Thaliet al., J. Virol., 65:6188-6193 ((1991)). These mutations includeresidue 257 mutated from threonine to arginine (257 T/R), 368 D/R, 370E/R, 457 D/A and 477 D/V. Most of these mutations abrogate Fab bindingor reduce it to low levels consistent with the assignment of therecombinant Fabs in this assay as reacting with the CD4 site.

In a particular mutant of gp120, the V1/V2 loop (residues 119-205) iscompletely removed. This mutation enhances the binding of Fabs b6, b11,and b14 but significantly decreases the binding of Fab b12. Deletion ofthe V3 loop produces a more modest decrease in Fab b12 binding whilegenerally enhancing the binding of the other Fabs. The 314 G/W change inthe V3 loop produces a decrease in binding of all the Fabs. This effecthas been observed for other CD4 binding site antibodies (Moore andSodroski, unpublished observations).

When the binding specificities of each Fab is examined in detail, eachFab has a unique mutant binding profile. For example, Fab b14 binding iseliminated by the 113 D/A change whereas the binding of the other Fabsis unchanged or enhanced; Fab b3 and b11 binding is reduced by the475M/S mutation but binding by the other Fabs is unchanged and the 370E/Q change reduces binding of all the Fabs except for b6 and possiblyb11. Fab b12 is distinguished by its decreased binding to a mutant inwhich the V1 and V2 loops are deleted. This may or may not be related tothe enhanced neutralizing ability of Fab b12 and will be the subject offurther study. However, it is clear that the V1 and V2 loops and the V3loop can affect antibody binding to the CD4 binding site either bydirect contact or transmitted conformational effects.

The effects on Fab binding of a series of point mutations in gp120afford the opportunity to look more closely at recognition differences.The general patterns observed are broadly reminiscent of many CD4 siteantibodies and of soluble CD4 itself. Fab b12 is distinguished by itsdecreased binding to a mutant in which the V1 and V2 loops are deleted.This may or may not be related to the enhanced neutralizing ability ofFab b12. It will be necessary to study a number of variants of Fab b12,which could be produced by chain shuffling or mutation, to answer thisquestion. However, it is clear that the V1 and V2 loops and the V3 loopcan affect antibody binding to the CD4 binding site either by directcontact or transmitted conformational effects.

6. Determination of the Relationship Between the Epitopes Recognized bythe Fabs with HIV-1 Antigen Multimeric Complexes

a. Comparison of Fab Epitopes with gp120 and gp160 Expressed asMultimeric Complexes on the Surface of COS-1 Cells

Given the lack of correlation of Fab neutralization with bindingparameters assessed using recombinant gp120, the binding of Fabs b3, b6,and b12 to COS-1 cells expressing the HXBc2 envelope glycoproteins gp160and gp120 was compared. Fab b3, the poorest neutralizer, Fab b6, also apoor neutralizer, and Fab b12, the most effective neutralizer asdetermined in Example 5 were used in the assay. The envelopeglycoproteins expressed by the COS-1 cells were gp160, the precursor ofgp120 and gp42, and the mature gp120. In this assay, differentconcentrations of Fab were incubated with radiolabeled COS-1 cells whichexpress gp160 and gp120 on their surface. The cells were then washed andlysed. The gp120 and gp160 envelope glycoproteins bound to Fab wereprecipitated with goat anti-F(ab')₂ antibody and analyzed by protein gelelectrophoresis and shown in FIG. 20. Since the amount of HIV-1 envelopeglycoprotein expressed on the surface of transfected COS-1 cells issmall compared with the amount present intracellularly, after celllysis, the bound Fab is presented with a large excess of both maturegp120 and gp160 precursor forms. The total amount of envelopeglycoproteins precipitated thus provides an indication of the amount ofFab bound to the cell surface. Scanning densitometry profiles werederived from the autoradiographs and are expressed in arbitrarydensitometric units.

Although the lack of saturation for Fabs b6 and b3 precludes a preciseestimate of affinity, it is clear that Fab b3 exhibits a lower affinityfor the precursor gp160 than either Fab b6 or b12. When the binding ofFab b12 and b6 are compared, several differences are apparent. Assumingthat Fab 6 achieves saturation at concentrations slightly higher than150 μg/ml, the estimated affinities of Fab b12 and b6 for the totalpopulation of envelope glycoproteins recognized differ only marginally.The most striking difference in the binding of Fab b12 and b6 to themultimeric envelope glycoprotein complex is the preferential detectionof gp120 relative to gp160 by Fab b12. Using densitometry to estimateamounts, it is seen from FIG. 20 that Fab b12 immunoreacts with anamount of gp120 that is at least about 50 % more than the gp160 presentin the immunoreaction admixture. The estimated affinities, based on theFab concentrations at which half-maximal binding to gp120 is observed,are 3×10⁷ M⁻¹ and <6×10⁶ M⁻¹ for Fabs b12 and b6, respectively.

The binding of the Fabs to the multimeric envelope glycoprotein complexon the transfected COS-1 cell surface provides some insights into theobserved differences in neutralization potency. The binding of the mostpotent neutralizing Fab, Fab b12, achieves saturation at roughly 100μg/ml, whereas neither of the less potent neutralizing Fabs achievessaturation even at 150 μg/ml. Fab b3 clearly exhibits a lower affinityfor the cell surface envelope glycoprotein complex than do the other twoFabs tested, b12 and b6. The most striking difference in the binding ofb12 and b6 to the multimeric envelope glycoprotein complex is thepreferential precipitation of gp120 relative to gp160 by the bound Fabb12. In addition to these differences in gp120 recognition, it appearsthat the overall number of cell surface envelope glycoproteins capableof being recognized by the less neutralizing Fabs is greater than thatseen for Fab b12. These differences suggest that Fab b12 may recognize amore limited subset of envelope glycoprotein conformations and thatthese conformations are better approximated by the mature gp120glycoprotein in the cell lysates. It is known that the gp160 precursorassumes a greater variety of conformations during the maturation processthan does the fully folded gp120 product (Thiriart, et al., J. Immunol.,143:1832-1836 (1989) and Fennie and Lasky, J. Virol., 63:639-646(1989)). The enhanced neutralization ability of Fab b12 could reflect ahigher affinity for a restricted gp120 conformation present in thefunctionally relevant subset of envelope glycoprotein spikes. Such afunctionally relevant group of envelope glycoproteins moieties probablyrepresents a small subset of the total population, consistent with thelow infectious fraction associated with HIV-1 and other retroviral viruspreparations. One caveat to these observations is that the glycosylationof gp120 expressed as a recombinant protein in baculovirus or on thesurface of COS-1 cells is likely to differ and this could affect bindingof the Fabs of this invention. However, no difference in the affinityfor CD4 binding site antibodies between the two forms of gp120 has beenobserved previously using a range of antibodies (Moore and Sodroski,unpublished observations). In addition, these studies employed amolecular clone of HIV-1 and its extension to primary isolates will needto be studied further.

Fabs derived from combinatorial libraries may be viewed as "artificial".However, as shown here, the recognition properties of a set ofantibodies directed to the CD4 site of gp120 show many features incommon with those derived by conventional means. They also show manyfeatures in common with one another suggesting that, with the caveatsinherent in the library approach (Barbas et al., J. Molec. Biol.,230:812-823 (1993) and Burton and Barbas, Nature, 359:782-783 (1992)),one individual produces several clearly distinct antibodies directed toa common structural feature, i.e., the CD4 binding site. This is inagreement with observations made on anti-CD4 binding site antibodiesusing anti-idiotype antibodies (Chamat et al., J. Immunol., 149:649-654(1992) and Hariharan et al., J. Virol., 67:953-960 (1993)). Oneadvantage of producing several antibodies is that escape (at least inbinding terms) is made more difficult. The only mutations in Table 9which essentially eliminate the binding of all the antibodies alsoreduce CD4 binding ability.

The observations presented here have significance for vaccinedevelopment. The most effective vaccine may need to induce antibodies tothe CD4 binding site with properties similar to those of Fab b12. Giventhe data above, recombinant gp120 offers no special qualities in thisregard. Further, the Fab b12 type of antibody formed only about 10%(4/33 Fabs) of the cloned response of the library donor (Barbas et al.,J. Molec. Biol., 230:812-823 (1993)) and has not been described amongstthe human antibodies derived by other means suggesting it may be a minorcomponent of typical responses. It is clearly of some interest forvaccine design to define more precisely the structure recognized by Fabb12.

7. Recognition of gp120 from Primary HIV-1 Isolates by b12 IgG1 in Vitro

The ability of the b12 IgG1 to recognize the gp120 molecule from HIV-1virus from 69 primary isolates was determined in an ELISA assay.Recognition of the primary HIV-1 virus isolate with b12 IgG1 isindicative of the prevalence of the b12 epitope in the HIV-1 pandemic.To probe the occurrence of the b12 epitope in the HIV-1 pandemic,binding of the b12 IgG1 to gp120 from 69 international isolatesbelonging to 6 different clades was examined. Virus isolates assayedwere obtained from the WHO, HMJFAMM, and NIAID.

Infectious culture supernatants containing virus and free gp120 weretreated with 1%(v/v) Nonidet-P40 (NP40) non-ionic detergent to provide asource of gp120 (Moore et al., AIDS, 3:155-160 (1989)). Microplate wells(Immulon II, Dynatech, Ltd.) were first coated with sheep polyclonalantibody D7324. This antibody was raised to the peptide APTKAKRRWQREKR,derived from the C-terminal 15 amino acids of the clade B IIIB HIV-1viral isolate. Next, an appropriate volume of inactivated supernatantcontaining gp120 was diluted with a buffer comprising tris-bufferedsaline (TBS)/1% (v/v) NP40/10% fetal calf serum (FCS) and a 100 μlaliquot added to the microplate wells for 2 hours at room temperature.Unbound gp120 was removed by washing with TBS, and bound gp120 wasdetected with CD4-IgG (1 μg/ml) or with b12 IgG1 diluted in a buffercomprising TBS/2%(w/v) nonfat dry milk powder/20%(v/v) sheet serum(TMTSS) essentially as previously described (Moore et al., AIDS,4:307-310 (1990)) and Moore et al., J. Virol., 68:469-473 (1994)).CD4-IgG is a fusion molecule which consists of CD4 and IgG. The CD4portion binds to gp120 and the IgG portion provides the means fordetection of the CD4-IgG fusion molecule with labeled anti-IgG reagents.Bound antibody was then detected with an appropriatealkaline-phosphatase conjugated anti-IgG, followed by AMPAK (DakoDiagnostics). Absorbance was determined at 492 nm (OD₄₉₂). Each viruswas tested against CD4-IgG in triplicate and against b12 IgG1 induplicate. All OD₄₉₂ values were corrected for non-specific antibodybinding in the absence of added gp120 (buffer blank). The mean,blank-corrected OD₄₉₂ values for CD4-IgG and b12 IgG1 were thencalculated, and the OD₄₉₂ ratios of b12 IgG1:CD4-IgG were determined.This normalization procedure enables allowance to be made for thedifferent amounts of gp120 captured onto the solid phase via antibodyD7324 when comparing antibody reactivity with a panel of viruses.Binding ratios of 0.50 or greater were deemed to represent strongantibody reactivity; ratios from 0.25-0.50 were considered indicative ofmoderate reactivity; values of <0.25 were designated as representativeof essentially negative monoclonal antibody reactivity.

As shown in FIG. 22, b12 IgG1 reacts with ≧50% of clades A-D but only 1of 12 isolates from clade E. Reactivity with clade B isolates from theU.S.A. is approximately 75%.

8. Nucleic Acid Sequence Analysis Comparison Between HIV-1 SpecificMonoclonal Antibody Fabs and the Corresponding Derived Amino AcidResidue Sequence

To explore the relationship between neutralizing and weakly ornon-neutralizing Fabs, the variable domains of 32 clones expressinghuman anti-gp120 Fabs, prepared in Example 2 including the 20 listed inFIG. 6 for which neutralizing activity was assessed, were sequenced. Inaddition, the five gp41-specific Fabs were also sequenced.

Nucleic acid sequencing was performed on double-stranded DNA usingSequenase 1.0 (USB, Cleveland, Ohio) and the appropriate primershybridizing to sequences in the Cg1 domain (SEQGb: 5'GTCGTTGACCAGGCAGCCCAG 3' SEQ ID NO 49) or the Ck domain (SEQKb: 5'ATAGAAGTTGTTCAGCAGGCA 3' SEQ ID NO 50). Alternatively sequencingemployed single stranded DNA and the T3 primer (5' ATTAACCCTCACTAAAG 3',SEQ ID NO 51) or one hybridizing to a sequence in the Ck domain (KEF: 5'GAATTCTAAACTAGCTAGTTCG 3' SEQ ID NO 52).

The amino acid residue sequences of the variable heavy and light chainsderived from the nucleic acid sequences of the 32 gp120-specific clonesare shown respectively in FIGS. 10 and 11. Groupings are made on thebasis of similarities in heavy chain sequences. Dots indicate identitywith the first sequence in each section. The SEQ ID NOs are listed tothe right of the corresponding derived heavy and light chain (V_(H) fromSEQ ID NO 53-81 and V_(L) from SEQ ID NO 82-113) amino acid residuesequences in the Figures themselves.

Alignment of derived sequences with one another and with the Genbankdatabase made use of the MacVector suite of programs. For analysis ofheavy chain CDR3 sequences as described by Sanz, J. Immunol.,147:1720-1729 (1991), the most 5' nucleotide was considered to be thefirst nucleotide after codon 95 of the H chain variable region accordingto Kabat et al, Sequences of Proteins of Immunological interest, USDept. of Health and Human Services, Washington, DC (1991). The most 3'nucleotide was assigned to the last unidentified nucleotide before thesequence matched with the published germline JH genes. The CDR3sequences were analyzed using the DNASTAR software. Sequence comparisonswere performed with both the ALIGN and COMPARE programs in order todetermine the germline D gene which provided the best homologythroughout. In a second step, the SEQCOMP program was used to findsequence identity of at least six nucleotides with either the codingstrand or the reverse complement of germline D genes.

The heavy and light chain sequences of the gp41-specific Fabs are shownin FIGS. 18 and 19, respectively. The amino acid residue sequence of theCDR3 heavy chain exhibits the most variation between the Fabs than anyother region of the variable domain.

a. Organization of Antibodies into Groups According to Heavy ChainSequence

V_(H) and V_(L) domains of 32 gp120 clones were sequenced and the V_(H)domains compared using MacVector software. This analysis immediatelyestablished that a number of the clones, including those selected bypanning against different antigens, are closely related to one another.The exception to this is the Fabs selected by panning against the V3loop peptide which are not related to the Fabs selected by panningagainst the gp120/160 antigens. FIG. 10 shows that the V_(H) sequencesderived from gp120/160 panning can be organized into 7 groups. The broadfeatures apparent from a comparison of amino acid sequences arediscussed herein.

The relatedness of sequences within a group varies considerably. Forinstance, in the group beginning with clone number b8 the amino acidsequences are very similar. Six clones were identical and the remaindershowed a maximum of 5 differences from the predominant sequence (the EQdifference due to the 5' primer excluded). Only one clone showed asingle difference in the CDR3 region. The average discrepancy over allthe sequences in this group from the predominant sequence is 1.1 aminoacid residues/variable domain. This amount corresponds to the order ofmagnitude of discrepancies which could arise from the PCR. Sequencing ofconstant domains indicated a PCR error frequency of about 1 base changeper domain.

In contrast, in the group headed by clone b3, no two clones wereabsolutely identical. The average difference from the consensus groupsequence is 3.3 residues per sequence and determination for the CDR3alone is 1.3. Therefore, it seems likely that the heavy chains in thisgroup are somatic variants of one another.

The group headed by clone 1 presents a third pattern. Clones b1 and b14are identical as are clones b2 and B2. However, 23 amino aciddifferences exist between the two sets of clones. Clones b24 and B30 areapproximately equally well differentiated (13-25 differences) fromeither of these two sets of clones or one another. Still the CDR3regions are very similar. A number of explanations can be suggested forthis pattern: 1) all clones in this group originate from the samegermline gene which has undergone extensive somatic mutation, 2)cross-over events have occurred to essentially recombine differentgermline genes with the same DJ combination, 3) a "convergent evolution"process has led to the selection of different germline genes associatedwith the same DJ combination.

b. Sequences of the V_(L) Domains from the gp120 Binders

The V_(n) sequences of the Fabs were organized into the groups definedin FIG. 10 are shown in FIG. 11. Immediately apparent was the extensivechain promiscuity as evidenced by the pairing of different light chainswith the same or a very similar heavy chain with retention of antigenbinding capability and indeed, for the most part, antigen affinity ascompared with FIG. 10. This promiscuity can be explored further byreference to the groups considered above.

The clone b8 group, in which the heavy chain members were identical orvery similar, also produced 4 light chains which are identical or verysimilar (less than 3 amino acid differences). Therefore a predominantheavy-light chain combination can be described for this group. Onemember (clone b8) had the same or very closely related V_(L) gene butappeared to use a different Jk gene. Two other members (clones B8 andb18) were more distantly related to the major sequence (7-12differences). Two further clones (b13 and B26) used a Vk gene from adifferent family, Vk3 compared to Vk1, and therefore were unrelated tothe major sequence.

The clone b3 group, suggested to contain somatic variants of a singleheavy chain, showed considerable light chain diversity with no twomembers being closely related to one another. Vk3-Jk2 combinationspredominated but Vk3-Jk3 and Vk1-Jk3 combinations also occurred.

On the other hand, in the clone b1 group evidence existed for the heavychains being more choosy about their light chain partner. Thus, closelyrelated heavy chains appeared to be paired with related light chains.The identical heavy chain pairs (b1 and b14; b2 and B2) had very similarlight chains (2 and 4 amino acid differences respectively) whereas thedistinct heavy chains (b24 and B30) had distinct light chains which wereunrelated to one another or the other group members. The clone 4 groupprovides another example of this phenomenon in that 4 closely relatedheavy chains were paired with 3 closely related light chains (apredominant heavy-light chain combination), except for the clone b7light chain that was distinct.

In summary, the heavy chain (V_(H)) sequences was organized into 7groups where each member of a group has an identical or very similarCDR3 region with a limited number of differences elsewhere. When thelight chains (V_(L)) were constrained into the groupings defined bytheir heavy chain partners, considerable light chain sequence variationwas observed. This phenomenon of chain promiscuity has been observedpreviously and can be appreciated by reference to FIG. 11. Markedneutralizing ability was confined to two groups of sequences. The firstgroup consisted of Fabs 4, 7, 12 and 21 which have very similar heavyand light chains. The second group consisted of Fabs 13, 8, 18, 22 and27. Only Fab 13 showed marked neutralizing ability, although the othersshowed some weaker activity. Interestingly in this group Fab 13 did havea light chain distinct from the other members of the group.

9. Shuffling of the Heavy and Light Chain of a Single Clone Against theLibrary

To further explore possible functional heavy-light chain combinations,the heavy chain of clone b12 (also referred to as Fab 12 for thecorresponding soluble Fab preparation) shown in FIG. 10 was recombinedwith the original light chain library prepared in Example 2 to constructa new library H12-LCn. In addition, the b12 light chain was recombinedwith the original heavy chain library to construct a library Hn-L12.These two libraries were taken through 3 rounds of panning against gp120(IIIB) as described in Example 2b5). The Fabs expressed from theresultant immunoreactant clones were analyzed as described in Example 3above. Clone b12 was chosen as this Fab neutralized HIV-1 in vitro asshown in Example 3.

To accomplish the preparation of a shuffled library from the Fd gene ofclone b12 with the original light chain library, the b12 heavy chain wasfirst subcloned into a tetanus toxoid binding clone expressed inpComb2-3. The light chain library was then cloned into this constructionto give a library of 1×10⁷ members. The subcloning step was used toavoid contamination with and over-representation of the original lightchain. A similar procedure was adopted for shuffling of heavy chainsagainst the light chain from clone b12 to give a library of 3×10⁶members. Cloning and panning procedures were carried out as describedabove for the original library.

Eleven light chains which recombined with the b12 heavy chain and boundgp120 by panning were randomly chosen for subsequent competition ELISAand sequence analysis. The apparent affinities of these shuffledcombinations were similar with an IC₅₀ of approximately 10⁻⁸ to 10⁻⁹ M.The sequences were organized where a set of 3 were very similar to theoriginal b12 light chain and the other 8 showing many differences fromthe original with some sub-grouping possible.

The sequences of the light chains which bound to the b12 heavy chainclone are shown in FIG. 12. The sequences are compared to the sequencefor the original light chain from clone b12. The light chains areidentified by numbers which do not correspond to the original lightchain clones; the assigned numbers of the newly selected clones havingnew light chains are thus arbitrary. The sequences of these light chainsare also listed in the Sequence Listing from SEQ ID NO 114 to 122. Somelight chain sequences are identical. In addition to immunoreactivitywith gp120, the new Fabs isolated from these shuffled clones were testedin the syncytia assay for neutralization of HIV-1 infection as describedin Example 3. Four shuffled monoclonal Fab antibodies, each having theheavy chain from clone b12, a known HIV-1 neutralizing clone, and newlight chains designated L28, L25, L26 and L22, all exhibitedapproximately 60% neutralization in a syncytia assay with 0.4 μg/mlpurified Fab. This effect was equivalent to that obtained with theoriginal clone b12 heavy and light chain pair. Maximum neutralization ofapproximately 80% was obtained with the H12/L28 and H12/L25 Fabs at 0.7μg/ml which was equivalent to that seen with the original clone b12heavy and light pair. The neutralization resulting from the H12/L22 andH12/L26 Fabs plateaued at 60% with Fab concentrations of 0.4 μg/ml up to1.0 μg/ml. Thus, in addition to the gp120 immunoreactive and HIVneutralizing Fabs obtained in the original library prepared as describedin Example 2, by shuffling a known neutralizing heavy chain with alibrary of light chains, new HIV-1 neutralizing Fab monoclonalantibodies have been obtained.

Ten heavy chains which recombined with the b12 light chain were alsorandomly chosen. One was very similar to the original b12 heavy chainbut the others have many differences. Nevertheless, the V-D and D-Jjunctions were essentially identical indicating the clones had probablyarisen from the same rearranged B-cell clone by somatic modification.Competition ELISA failed to reveal any clear difference in affinitybetween the variants selected from those originally analyzed.

The sequences of the heavy chains which bound to the b12 light chainclone are shown in FIG. 13. The sequences are compared to the sequencefor the original heavy chain from clone b12. The heavy chains areidentified by numbers which do not correspond to the original lightchain clones; the assigned numbers of the newly selected clones havingnew heavy chains are thus arbitrary. The sequences of these light chainsare also listed in the Sequence Listing from SEQ ID NO 123 to 132. Somelight chain sequences are identical. In addition to immunoreactivitywith gp120, the new clones were tested in the syncytia assay forneutralization of HIV-1 infection as described in Example 3. Twoshuffled monoclonal Fab antibodies, each having the light chain fromclone b12, a known HIV-1 neutralizing clone, and new heavy chainsdesignated H2 and El4, exhibited approximately 40% neutralization in asyncytia assay with 1.0 and 0.5 μg/ml purified Fab, respectively. Thiseffect was equivalent to that obtained with the original clone b12 heavyand light chain pair at a concentration of 2 μg/ml. Maximumneutralization of approximately 50% was obtained with the Fab having thenew H14 chain at 1.0 μg/ml compared to 80% neutralization with 0.7 μg/mlwith the original clone b12 heavy and light pair. Thus, in addition tothe gp120 immunoreactive and HIV neutralizing Fabs obtained in theoriginal library prepared as described in Example 2, by shuffling aknown neutralizing light chain with a library of heavy chains, new HIV-1neutralizing Fab monoclonal antibodies have been obtained.

Thus, this shuffling process revealed many more heavy and light chainpartners that bound to gp120 that were equal in affinity to thoseobtained from the original library prepared in Example 2. With thisapproach, additional HIV-1 neutralizing antibodies can easily beobtained over those present in an original library. The complexity ofthe clones arising from the heavy chain shuffling also suggests thatthis approach may be used to map the course of somatic diversification.

Combinatorial libraries randomly recombine heavy and light chains so towhat extent antibodies derived from such libraries represent thoseproduced in a response in vivo can be determined. In principle, aheavy-light chain combination binding antigen could arise fortuitously,i.e., neither chain is involved in binding antigen in vivo but thecombination does bind antigen in vitro.

The available data suggests, however, that heavy chains, from immunelibraries, involved in binding antigen tightly in vitro arise fromantigen-specific clones in vivo. First, studies have generally failed toidentify high-affinity binders in non-immunized libraries. See, Perssonet al. Proc. Natl. Acad. Sci., USA, 88:2432-2436 (1991) and Marks et al.Eur. J. Immunol., 21:985-991 (1991).

Further, as described above, gp120 binders were not observed in panninga bone marrow IgG library from an HIV seronegative donor against gp120.Second, heavy chains associated with binders from immunized librarieswere typically at relatively high frequency in the library indicatingthey were strongly represented in the mRNA isolated from immunizedanimals. See, Caton et al., Proc. Natl. Acad. Sci., USA, 87:6450-6454(1990) and Persson et al., supra. Third, heavy chains from immunizedlibraries appeared to dictate specificity when recombined with variousunrelated light chains as described in Example 10. Fourth, the isolationof intraclonal heavy chain variants as here indicated that an activeantibody response was cloned. Thus, the shuffling of a known heavy chainwith a light chain binder and vice versa is preferred for use in thisinvention as new neutralizing Fabs can be obtained beyond thosegenerated in vivo.

Heavy chain promiscuity, i.e., the ability of a heavy chain to pair withdifferent light chains with retention of antigen affinity, presentsserious problems for identifying in vivo light chain partners. Thisapplies not only to the strict definition of partners as having arisenfrom the same B-cell but also to one which would encompass somaticvariants of either partner. The existence of predominant heavy-lightchain combinations, particularly involving intraclonal light chainvariants, suggests that the light chains concerned are well representedin the library and probably are associated with antigen binding in vivo.However, promiscuity means that, although some combinations probably dooccur in vivo, one cannot be certain that one is not shuffling immunepartner chains in the recombination. For instance, the occurrence of avirtually identical light chain (b6, B20) in 2 out of 33 clones suggeststhat it is probably over-represented in the library consistent with anin vivo involvement in antigen-stimulated clones. However, there is noway of knowing whether the in vivo partner of the light chain is the b6or B20 heavy chain or indeed another heavy chain arising from astimulated clone.

The light chains arising from the combinatorial library may not be thoseemployed in vivo. Nevertheless it is interesting to note that some heavychains appear relatively choosy about light chain partner whereas othersappear almost indifferent. This observation needs to be tempered by thefinding that apparently choosy heavy chains from this analysis willaccept diverse light chains with maintenance of antigen binding in abinary plasmid system where pairings are forced as shown below inExample 11 rather than selected in a competitive situation.

Two reports compare heavy-light chain combinations arising fromcombinatorial libraries and hybridomas in immunized mice. The libraryapproach begins with mRNA and is therefore probably reflecting plasmacell populations. In contrast, hybridomas are thought to reflectactivated but not terminally differentiated B cell populations and EBVtransformation to reflect resting B cell populations.

Whatever the arguments about light chain authenticity, the heavy chainsof FIG. 10 present many features of interest. The most frequently usedheavy chain is of the clone b8 type. It could be argued that this usagesimply represents bias in PCR amplification. However, the occurrence ofapproximately equal numbers of clones in this group amplified by VH1aand VH3a primers argues against this notion. Furthermore, the existenceof intraclonal variants in some groups indicates that one is at leastsampling different genes from the initial library.

The antibodies cloned here do bear qualitative relationship with thepolyclonal antibodies present in the serum of the asymptomatic donor.The titer of anti-gp120 (IIIB) antibodies was approximately 1:3000, withgreater than 50% of the reactivity being inhibited by CD4 or a cocktailof Fabs from clones 12, 13 and 14. The titer of anti-gp120 (SF2)antibodies was approximately 1:800. Further, the titer of serum againstthe short constrained V3 loop peptide was 1:500 and against the fulllength MN V3 loop peptide was only 1:300. The importance of "anti-CD4site antibodies" seems general in donors with longer term HIV infectionin that the cocktail of Fabs 12, 13 and 14 was able to inhibit bindingof a large fraction of serum antibody reactivity with gp120 (IIIB) in 26of 28 donors tested.

The ability of Fabs to neutralize viruses has been a controversial area.One of the problems has been that Fabs are classically generated bypapain digestion of IgG. If the Fab, as is often the case, shows reducedactivity relative to the parent IgG then it may be difficult to rule outIgG contamination in the Fab preparation. Recombinant Fabs, however, asshown herein definitively neutralize virus.

The mechanism of neutralization of HIV-1 appears to neither requirevirion aggregation nor gp120 cross-linking. In addition, there is nocorrelation with blocking of the CD4-gp120 interaction toneutralization. The existence of the cloned neutralizing Fabs of thisinvention should allow the molecular features that confer neutralizingpotential to be explored. For instance, in the case of the group ofclones containing Fab 13, the unique character of the light chain ofthat neutralizing clone suggests that chain shuffling experiments inwhich the 13 light chain was recombined with the other heavy chains inthat group, might be revealing. Heavy chains paired with two dissimilarlight chains have been shown to retain antigen affinity but exhibitaltered fine specificity as shown in Example 11.

The observation here of a large number of Fabs with only a limitednumber being strongly neutralizing may have important consequences. Ifthe pattern is repeated for whole antibodies then it would seem thatmuch of the gp120 structure may be in a sense a "decoy", i.e., theimmune system may invest considerable effort in producing antibodies ofhigh affinity but limited anti-viral function. To exacerbate thesituation the ineffective antibodies may bind to gp120 and inhibit thebinding of strongly neutralizing antibodies. This has obviousconsequences for vaccination which should be primarily designed toelicit neutralizing antibodies of this invention.

10. Shuffling of Selected Heavy and Light Chain DNA Sequences of aCombinatorial Library in a Binary Plasmid System

A binary system of replicon-compatible plasmids has been developed totest the potential for promiscuous recombination of heavy and lightchains within sets of human Fab fragments isolated from combinatorialantibody libraries. The efficiency of the system is demonstrated for thecombinatorial library of this invention derived from the bone marrowlibrary of an asymptomatic HIV donor.

a. Construction of the Binary Plasmid System

The binary plasmids pTAC01H and pTC01 for use in this invention containthe pelB leader region and multiple cloning sites from Lambda Hc2 andLambda Lc3, respectively, and the set of replicon-compatible expressionvectors pFL281 and pFL261. Both pFL281 and pFL261 have been described byLarimer et al., Prot. Eng., 3:227-231 (1990), the disclosure of which ishereby incorporated by reference. The nucleotide sequences of pFL261 andpFL281 are in the EMBL, GenBank and DDBJ Nucleotide Sequence Databasesunder the accession numbers M29363 and M68946. The plasmid pFL281 isbased on the plasmid pFL260 also described by Larimer et al., supra, andhaving the accession number M29362. The only distinction between theplasmids pFL260 and pF1281 is that pFL281 lacks a 60 bp sequence ofpFL260 between the Eag I site and the Xma III site resulting in the lossof one of the two BamH I sites. This deletion is necessary to allow forcloning of the BamH I Hc2 fragment into the expression vector asdescribed herein.

The replicon-compatible expression vectors share three common elements:(i) the f1 single-stranded DNA page intergenic IG regions; (ii) thetightly regulated tac promoter and lac operator; and (iii) an rbs-ATGregion with specific cloning sites. The plasmid vectors differ in theirantibiotic resistance markers and plasmid replicons: pFL261 carries agene encoding chloramphenicol acetyltransferase (cat), conferringchloramphenicol resistance, and the p15A replicon; pFL281 carries a geneencoding beta-lactamase (bla), conferring ampicillin resistance, and theColE1 replicon (ori) from pMB1. The p15A and ColE1 replicons permit thecoincident maintenance of both plasmids in the same E. coli host.

The Hc2 and Lc2 vectors prepared in Examples 1a2) and 1a3),respectively, were converted into the plasmid form using standardmethods familiar to one of ordinary skill in the art and as described bySambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, New York (1989) and subsequentlydigested with Xho I-Spe I (pHc2) and Sac I-Xba I for (pLc2). Thesynthetic linkers for insertion into the digested pHc2 and Lc2 plasmidswere prepared by American Synthesis. The linkers were inserted toincrease the distance between cloning sites so as to increase theeffectiveness of the digestions. The 5' and 3' linkers for preparing thedouble-stranded linker insert into pHc2 were 5'TCGAGGGTCGGTCGGTCTCTAGACGGTCGGTCGGTCA 3' (SEQ ID NO 133) and 5'CTAGTGACCGACCGACCGTCTAGAGACCGACCGACCC 3' (SEQ ID NO 134), respectively.The 5' and 3' linkers for preparing the double-stranded linker insertinto pLc2 were 5' CGGTCGGTCGGTCCTCGAGGGTCGGTCGGTCT 3' (SEQ ID NO 135)and 5' CTAGAGACCGACCGACCCTCGAGGACCGACCGACCGAGCT 3' (SEQ ID NO 136),respectively. The pairs of linker oligonucleotides were separatelyligated to their respective digested, calf intestinalphosphatase-treated vectors.

Subsequently, the multiple cloning sites of pHc2 and pLc2 weretransferred into the expression vectors, pFL281 and pFL261,respectively. To accomplish this process, the multiple cloning regionsof both Lc2 and Hc2 were separately amplified by PCR as described byGram et al., Proc. Natl. Acad. Sci., USA, 89:3576-3580 (1992) and asdescribed in Example 2b using Vent Polymerase (New England Biolabs)according to the manufacturer's recommendations. The forward primer, 5'CAAGGAGACAGGATCCATGAAATAC 3' (SEQ ID NO 137) was designed to provide aflush fusion of the pelB leader sequence to the ribosome binding sitesof the cloning vectors' pFL261 and pFL281 via its internal BamH I siteindicated by the underlined nucleotides. The reverse primer 5'AGGGCGAATTGGATCCCGGGCCCCC 3' (SEQ ID NO 138) was designed to annealdownstream of the region of interest in the parent vector of pHc2/pLc2and create a second BamH I site. The resultant Hc2 and Lc2 PCRamplification products were then digested with BamH I to provide forBamH I overhangs for subsequent ligation into BamH I linearized pFL281and pFL261 vectors, respectively. The resulting light chain vectorcontaining the Lc2 insert, designated pTC01, was used in this form,whereas the heavy chain vector was further modified with a histidinetail to allow purification of Fab fragments by immobilized metalaffinity chromatography as described by Skerra et al., Bio/Technology,9:273-278 (1991). For this purpose, the synthetic linkeroligonucleotides, respectively the 5' and 3' linkers, 5'CTAGTCATCATCATCATCATTAAGCTAGC 3' (SEQ ID NO 139) and 5'CTAGGCTAGCTTAATGATGATGATGATGA '3 (SEQ ID NO 140) was inserted into theSpe I site, in effect removing the decapeptide tag sequence to generatethe heavy chain vector designated as pTAC01H. The expression of Fabfragment in all subsequent cloning experiments was suppressed by adding1% (w/v) glucose to all media and plates.

b. Construction of Expression Plasmids

For expression of the light chain variable domain, pTC01 prepared abovewas first digested with Sac I and Xba I; individual light chain insertswere then obtained by separately digesting 22 of the pComb2-3 plasmidsprepared and screened as described in Example 2 and listed in FIG. 7that bind to gp120 with the same combination of enzymes and isolatingthe 0.7 kb fragment using low melting point agarose gel electrophoresisfollowed by b-agarose digestion. For the chain-shuffling experiments,the following representative members of each of the seven groups shownin FIG. 7 were chosen: b11; b6; b4-b12-b7-b21; b3; s8; b1-b14-b24;b13-b22-B26-b8-b18-b27-B8-B35-s4; and one loop peptide-binding clone,p35. The different groups are indicated by semicolon separations whilemembers of the same group are dashed. The resultant isolated lightchains were separately ligated into PTCO1 overnight at 16° C. understandard conditions using a 5:1 molar insert-to-vector ratio to form 21light chain pTCO1 expression vectors. For expression of the heavy chainvariable domain, pTAC01H prepared above was first digested with Xho Iand Spe I; heavy chain inserts were then obtained by separately PCRamplification reactions of the 20 pComb2-3 plasmids from which lightchain inserts were obtained. PCR was used to isolate the heavy chaininserts instead of restriction digestion in order to obtain heavy chainwithout the cpIII gene anchor sequence in the vector. For the PCRreaction, the respective 5' and 3' primers, 5' CAGGTGCAGCTCGAGCAGTCTGGG3' (VH1a) (SEQ ID NO 42) and 5' GCATGTACTAGTTTTGTCACAAGATTTGGG 3' (CG1z)(SEQ ID NO 44) were used to amplify the region corresponding to theheavy chain as described in Examples 2a1) and 2a2). The resultant PCRproducts were purified by low-melting point electrophoresis, digestedwith Xho I and Spe I, re-purified, and separately ligated to thesimilarly prepared heavy chain pTAC01H vector using a 1:2 molarvector-to-insert ratio to form 21 heavy chain pTACO1H expressionvectors.

c. Co-transformation of Binary Plasmids

CaCl₂ -competent XL1-Blue cells (Stratagene; recA1, endA1, gyrA96, thi,hsdR17, supE44, relA1, lac, {F' proAB, lacI^(q), ZDM15, Tn10(tet^(R))})were prepared and transformed with approximately 0.5 μg purified DNA ofeach plasmid in directed crosses of each of the 20 light chain vectorswith each of the 20 heavy chain vectors. The presence of both plasmidsand the episome was selected for by plating transformants ontriple-antibiotic agar plates (100 μg/ml carbenicillin, 30 μg/mlchloramphenicol, 10 μg/ml tetracycline, 32 g/l LB agar) containing 1%glucose.

A binary plasmid system consisting of two replicon-compatible plasmidswas constructed as shown in 14. The pTAC01H heavy chain vector schematicis shown in FIG. 14A and the pTCO1 light chain vector schematic is shownin FIG. 14B. Both expression vectors feature similar cloning sitesincluding pel B leader sequences fused to the ribosome binding sites andthe tac promoters via BamH I sites as shown in FIG. 15. The nucleotidesequences of the multiple cloning sites along with the tac promoter,ribosome binding sites (rbs) and the underlined relevant restrictionsites for the light chain vector, pTCO1, and heavy chain vector,pTAC01H, are respectively shown in FIG. 15A and FIG. 15B. The sequencesare also listed in the Sequence Listing as described in the BriefDescription of the Drawings. The heavy chain vector pTAC01H alsocontains a (His)₅ -tail to allow purification of the recombinant Fabfragments by immobilized metal affinity chromatography. The presence ofboth plasmids in the same bacterial cell is selected for by the presenceof both antibiotics in the media. Expression is partially suppressedduring growth by addition of glucose and induced by the addition of IPTGat room temperature. Under these conditions, both plasmids are stablewithin the cell and support expression of the Fab fragment as assayed byELISA using goat anti-human kappa and goat anti-human IgG1 antibodies.

d. Preparation of Recombinant Fab Fragments

Bacterial cultures for determination of antigen-binding activity weregrown in 96 well-tissue culture plates (Costar #3596). 250 μl Superbroth[SB had the following ingredients per liter: 10 g 3-(N-morpholino)propanesulfonic acid, 30 g tryptone, 20 g yeast extract at pH 7.0 at 25°C.) containing 30 μg/ml chloramphenicol, 100 μg/ml carbenicillin, and 1%(w/v)] glucose were admixed per well and inoculated with a singledouble-transformant prepared in Example 11c above. The inoculated plateswere then maintained with moderate shaking (200 rpm) on a horizontalshaker for 7-9 hours at 37° C., until the A₅₅₀ was approximately 1-1.5.The cells were collected by centrifugation of the microtiter plate(1,500× g for 30 minutes at 4° C.), the supernatants were discarded, andthe cells were resuspended and induced overnight at room temperature infresh media containing 1 mM IPTG, but no glucose. Cells were harvestedby centrifugation, resuspended in 175 μl PBS (10 mM sodium phosphate,160 mM NaCl at pH 7.4 at 25° C.) containing 34 μg/mlphenylmethylsulfonyl fluoride (PMSF) and 1.5% (w/v) streptomycinsulfate, and lysed by 3 freeze-thaw cycles between -80° C. and 37° C.The resultant crude extracts were partially cleared by centrifugation asabove before analysis by antigen-binding ELISA.

e. Assay and Determination of Relative Affinities

Relative affinities were determined as described in Example 266) aftercoating wells with 0.1 μg of antigen. The selected antigens includedtetanus toxoid and recombinant gp120 (strain IIIB) and gp120 (strainSF2). For each antigen, a negative control extract of XL1-Blue cellsco-transformed with pTC01 and pTAC01H was tested to determine whetherother components in E. coli had any affinity for the antigens in theassay. Each extract was assayed for BSA-binding activity andBSA-positive clones were considered negative. All possiblesingle-transformants expressing one chain only were prepared asdescribed for the double-transformants and were found to have noaffinity for any of the antigens used. Because of the nature of theassay, whether this was due to a lack of binding by the individualchains itself or due to a lack of expression or folding could not bedetermined.

f. Results of Direct Crosses of Heavy and Light Chains within a Set ofgp120/gp160 Binding Antibodies

The Fab fragments derived from the bone marrow of the same asymptomaticHIV donor but panned against gp120 (IIIB), gp160 (IIIB), and gp120(SF2), were assigned to one of seven groups based on the amino acidsequences of the CDR3 of their heavy chains as described in Example 9.From the same library, antibodies to the constrained hypervariablev3-loop-like peptide JSISIGPGRAFYTGZC (SEQ ID NO 141) were isolated. Forthe chain-shuffling experiments, the following representative members ofeach of the seven groups shown in FIG. 7 were chosen: b11; b6;b4-b12-b7-b21; b3; s8; b1-b14-b24; b13-b22-B26-b8-b18-b27-B8-B35-s4; andone loop peptide-binding clone, p35. Clones b4, b7, b12, and b21 showedneutralization activity against HIV when monitoring inhibition ofinfection by syncytia formation and clones b13, b12, and b4 whenmonitoring p24 production as shown in Example 3. Light and heavy chainswere cloned from the original constructs and cotransformed in allpossible binary combinations into XL1-Blue cells as described above.

The results of the complete cross are shown in FIG. 16. As is to beexpected, identical chains derived from different Fab fragments hadsimilar binding properties e.g., b18HC, b27HC, B8HC, B35HC, s4HC. Thecrosses of the original heavy chains with the original light chains ineach case clearly recapitulated binding activity. Minor differencesexisted between some heavy chains with identical variable domainsequences, e.g., b4 and b12 (constant domains were not sequenced for anyof the constructs). The exception is b8HC, which was identical in itsvariable domain to b18HC, b27HC, B8HC, B35HC, s4HC, yet shows more crossreactivity. Presumably, this is due to differences in expression levelsin the cell or differences in the constant domain sequences. Cleardifferences existed between heavy chains in their tendency to acceptdifferent light chains and still bind antigen, but even the leastpromiscuous heavy chain in the set panned against gp120 (IIIB), b1HC,still did so in 43% of its crosses. On the other side of the spectrum, 5heavy chains, b11HC, b6HC, b12HC, b7HC, and b8HC, crossed productivelywith all light chains in this set. For the heavy chain crosses examinedin detail (all of s4HC, B35HC, B26HC; most of b12HC, b12HC), nosignificant differences in apparent binding affinity were found betweenFab fragments using the same heavy chain but different light chains asshown in FIG. 17 where the IC₅₀ from competition with soluble gp120(IIIB) was approximately 10⁻⁸ M.

Within the original seven groups that were established according to thesequence of the CDR3 of the heavy chains and that are indicated byhorizontal and vertical lines in FIG. 16, complete promiscuity waspresent, i.e., heavy and light chains within these CDR3-determinedgroups were completely promiscuous with each other. However, there was alack of promiscuity between other groups, e.g., between b1HC-b24HC andb13LC-s4LC. In the analysis of these sequence-based groups, the proteinantigen against which the phage display library was panned was not acritical factor. The exception to this case was the cross of p35HC withall light chains; the only cross that bound either to gp120 (SF2 strain)or the original antigen, the loop peptide, was the cross containing theoriginal heavy and light chains.

Unlike the heavy chains, no light chains crossed productively with allheavy chains nor were any distinguishable from the other light chains byunusually low promiscuity.

In the neutralization assays performed as described in Example 3, thedirected cross resulting from the pairing of the heavy chain from cloneb12 with the light chain from clone b21, was effective at neutralizingHIV-1.

g. Interantigenic Crosses of Heavy and Light Chains

To determine whether conclusions derived from the crosses between highaffinity Fab fragments originating from the same library can be extendedto unrelated libraries, a non-related gamma1k-Fab fragment (P3-13)specific for tetanus toxoid from a different donor was chosen for a newset of crosses [clone 3 in Persson et al., Proc. Natl. Acad. Sci., USA,88:2432-2436 (1991)]. Extracts were probed with tetanus toxoid or withgp120 (IIIB). The data confirm the results from the gp120 crossexperiment in that the binding activity towards the antigen wasdetermined by the heavy chain. The heavy chain of clone P3-13 pairedwith the light chains b4, b12, b21, and b14 to yield an Fab fragmentwith an affinity towards tetanus toxoid; the light chain of P3-13 pairedwith the heavy chains of b3, b6, b11, and b14 to yield an Fab fragmentwith an affinity towards gp120 (IIIB). None of the light chainsoriginating from the gp120 binders was able to confer gp120 specificityin combination with the P3-13 heavy chain.

Similarly, the P3-13 light chain was unable to generate tetanus toxoidspecificity in combination with any of the heavy chains originating fromthe gp120 binders, confirming the dominance of the heavy chain in theantibody-antigen interaction. Interestingly, all three light chains thatshowed a strong signal against tetanus toxoid (b4, b12, b21) weremembers of the same group when sorted by the CDR3's of their originalheavy chains. As might be expected from crosses between unrelatedlibraries, not only was there a lower degree of promiscuity, i.e.,chains paired productively with far fewer complementary chains, but therange of apparent affinity constants determined by competition ELISA wasmuch broader (6.3×10⁶ -6.3×10⁻⁸ M). The replacement of the originalP3-13 light chain in the P3-13 Fab fragment with another light chainlowered the affinity of the Fab towards tetanus toxoid 10 to 100-fold(from 6.3×10⁻⁸ M to 6.3×10⁻⁸ M). In the crosses of the light chain ofP3-13 with all the heavy chains of the HIV pannings, the productivecrosses had similar affinities to gp120 (IIIB) (2.5×10⁷ -6.3×10⁻⁷ M),with the exception of b14HC/P3-13LC, whose signal was too weak for adefinite determination of the apparent binding constant. Theseaffinities were approximately five-fold lower than those of thegp120-heavy chains with their original light chains.

Thus, the results show that chain shuffling is yet another maneuverallowed in vitro but not in vivo which can be expected to help extendantibody diversity beyond that of Nature. The overriding feature of thebinary system of this invention is its ability to create large numbers(several hundred) of directed crosses between characterized light andheavy chains without the need for recloning individual chains for eachcross after the initial vector construction. When used in combinationwith the phage-display method and biological assays, it allows the rapidanalysis of the most interesting subset of the pool of antigen-bindingclones by chain shuffling, with the aim of finding biologically orchemically active antibodies. For the set of antigens studied here, mostheavy chains recombined with a number of light chains to yield anantigen-binding Fab fragment.

These results have important implications for the diversity ofcombinatorial antibody libraries. While it is not possible to predictreliably the original in vivo combinations of light and heavy chains dueto the surprising promiscuity of individual chains, recombinant antibodylibraries take advantage of the fact that even distantly related Fabsagainst the same antigen can recombine in vitro to give chaincombinations not found in vivo. In fact, after the identification of acertain number of antibodies that have been shown to possess somebiological or chemical activity, it may be better to shuffle theirindividual chains in a directed fashion than to continue samplingrandomly from the same pool of binders. By extension, the promiscuityobserved in this system indicates that in libraries constructed usingdegenerate, chemically synthesized oligonucleotides, there should beconsiderable flexibility in which separate synthetic heavy chains canpair with separate synthetic light chains to generate separateantigen-binding Fab fragments. The diversity of combinatorial librariescoupled with chain-shuffling should allow wide exploration of threedimensional space thereby solving the problem of how to approximatemolecules in the ternary complex of antibody, substrate and cofactor.

11. Deposit of Materials

The following cell lines have been deposited on Sep. 30, 1992, with theAmerican Type Culture Collection (ATCC), 1301 Parklawn Drive, Rockville,Md., USA:

    ______________________________________    Cell Line     ATCC Accession No.    ______________________________________    E. coli MT11  ATCC 69078    E. coli MT12  ATCC 69079    E. coli MT13  ATCC 69080    ______________________________________

The deposits listed above, MT11, MT12 and MT13 are bacterial cells (E.coli) containing the expression vector pComb2-3 for the respectiveexpression of the Fabs designated b11 (clone b11), b12 (clone b12), andb13 (clone b13) prepared in Example 2b. The sequences of the heavy andlight chain variable domains are listed in FIGS. 10 and 11,respectively. This deposit was made with the ATCC under the provisionsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purpose of Patent Procedure and theRegulations thereunder (Budapest Treaty). This assures maintenance of aviable culture for 30 years from the date of deposit. The organisms willbe made available by ATCC under the terms of the Budapest Treaty whichassures permanent and unrestricted availability of the progeny of theculture to the public upon issuance of the pertinent U.S. patent or uponlaying open to the public of any U.S. or foreign patent application,whichever comes first, and assures availability of the progeny to onedetermined by the U.S. Commissioner of Patents and Trademarks to beentitled thereto according to 35 U.S.C. §122 and the Commissioner'srules pursuant thereto (including 37 CFR §1.14 with particular referenceto 886 OG 638). The assignee of the present application has agreed thatif the culture deposit should die or be lost or destroyed whencultivated under suitable conditions, it will be promptly replaced onnotification with a viable specimen of the same culture. Availability ofthe deposited strain is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cell lines deposited,since the deposited embodiment is intended as a single illustration ofone aspect of the invention and any cell lines that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial does not constitute an admission that the written descriptionherein contained is inadequate to enable the practice of any aspect ofthe invention, including the best mode thereof, nor is it to beconstrued as limiting the scope of the claims to the specificillustration that it represents. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 170    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 173 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GGCCGCAAATTCTATTTCAAGGAGACAGTCATAATGAAATACCTATTGCCTACGGCAGCC60    GCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCCAGGTGAAACTGCTCGAGATT120    TCTAGACTAGTTACCCGTACGACGTTCCGGACTACGGTTCTTAATAGAATTCG173    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 173 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    TCGACGAATTCTATTAAGAACCGTAGTCCGGAACGTCGTACGGGTAACTAGTCTAGAAAT60    CTCGAGCAGTTTCACCTGGGCCATGGCTGGTTGGGCAGCGAGTAATAACAATCCAGCGGC120    TGCCGTAGGCAATAGGTATTTCATTATGACTGTCTCCTTGAAATAGAATTTGC173    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 131 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    TGAATTCTAAACTAGTCGCCAAGGAGACAGTCATAATGAAATACCTATTGCCTACGGCAG60    CCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGAGCTCGTCAGTTCTAGAG120    TTAAGCGGCCG131    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 139 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    TCGACGGCCGCTTAACTCTAGAACTGACGAGCTCGGCCATGGCTGGTTGGGCAGCGAGTA60    ATAACAATCCAGCGGCTGCCGTAGGCAATAGGTATTTCATTATGACTGTCTCCTTGGCGA120    CTAGTTTAGAATTCAAGCT139    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    TyrProTyrAspValProAspTyrAlaSer    1510    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    MetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAla    151015    AlaGlnProAlaMetAlaGlnValLysLeu    2025    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    MetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAla    151015    AlaGlnProAlaMetAlaGlu    20    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 198 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: circular    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    TGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCA60    CACAGGAGGAAGGATCCATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTAC120    TCGCTGCCCAACCAGCCATGGCCGAGCTCGGTCGGTCGGTCCTCGAGGGTCGGTCGGTCT180    CTAGAGTTAAGCGGCCGC198    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 198 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: circular    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GCGGCCGCTTAACTCTAGAGACCGACCGACCCTCGAGGACCGACCGACCGAGCTCGGCCA60    TGGCTGGTTGGGCAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAGGTATTTCA120    TGGATCCTTCCTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCC180    GATGATTAATTGTCAACA198    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    MetLysThrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAla    151015    AlaGlnProAlaMetAlaGluLeu    20    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 220 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: circular    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    TGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCA60    CACAGGAGGAAGGATCCATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTAC120    TCGCTGCCCAACCAGCCATGGCCCAGGTGAAACTGCTCGAGGGTCGGTCGGTCTCTAGAC180    GGTCGGTCGGTCACTAGTCATCATCATCATCATTAAGCTA220    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 220 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: circular    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    TAGCTTAATGATGATGATGATGACTAGTGACCGACCGACCGTCTAGAGACCGACCGACCC60    TCGAGCAGTTTCACCTGGGCCATGGCTGGTTGGGCAGCGAGTAATAACAATCCAGCGGCT120    GCCGTAGGCAATAGGTATTTCATGGATCCTTCCTCCTGTGTGAAATTGTTATCCGCTCAC180    AATTCCACACATTATACGAGCCGATGATTAATTGTCAACA220    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    MetLysThrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAla    151015    AlaGlnProAlaMetAlaGlnValLysLeuLeuGlu    2025    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: C-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    ThrSerHisHisHisHisHis    15    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GGCCGCAAATTCTATTTCAAGGAGACAGTCAT32    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    AATGAAATACCTATTGCCTACGGCAGCCGCTGGATT36    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    GTTATTACTCGCTGCCCAACCAGCCATGGCCC32    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    CAGTTTCACCTGGGCCATGGCTGGTTGGG29    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    CAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAG40    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    GTATTTCATTATGACTGTCTCCTTGAAATAGAATTTGC38    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    AGGTGAAACTGCTCGAGATTTCTAGACTAGTTACCCGTAC40    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    CGGAACGTCGTACGGGTAACTAGTCTAGAAATCTCGAG38    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    GACGTTCCGGACTACGGTTCTTAATAGAATTCG33    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    TCGACGAATTCTATTAAGAACCGTAGTC28    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    TGAATTCTAAACTAGTCGCCAAGGAGACAGTCAT34    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    AATGAAATACCTATTGCCTACGGCAGCCGCTGGATT36    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    GTTATTACTCGCTGCCCAACCAGCCATGGCC31    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    GAGCTCGTCAGTTCTAGAGTTAAGCGGCCG30    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 48 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    GTATTTCATTATGACTGTCTCCTTGGCGACTAGTTTAGAATTCAAGCT48    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    CAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAG40    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    TGACGAGCTCGGCCATGGCTGGTTGGG27    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    TCGACGGCCGCTTAACTCTAGAAC24    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 666 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    CCATTCGTTTGTGAATATCAAGGCCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTC60    AATGCTGGCGGCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGTGGCTCTGAG120    GGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCC180    GGTGATTTTGATTATGAAAAGATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCC240    GATGAAAACGCGCTACAGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTAC300    GGTGCTGCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCT360    ACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCA420    CCTTTAATGAATAATTTCCGTCAATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGC480    CCTTTTGTCTTTAGCGCTGGTAAACCATATGAATTTTCTATTGATTGTGACAAAATAAAC540    TTATTCGGTGTCTTTGCGTTTCTTTTATATGTTGCCACCTTTATGTATGTATTTTCTACG600    TTTGCTAACATACTGCGTAATAAGGAGTCTTAATCATGCCAGTTCTTTTGGGTATTCCGT660    TATTAT666    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 211 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    ProPheValCysGluTyrGlnGlyGlnGlyGlnSerSerAspLeuPro    151015    GlnProProValAsnAlaGlyGlyGlySerGlyGlyGlySerGlyGly    202530    GlySerGluGlyGlyGlySerGluGlyGlyGlySerGluGlyGlyGly    354045    SerGluGlyGlyGlySerGlyGlyGlySerGlySerGlyAspPheAsp    505560    TyrGluLysMetAlaAsnAlaAsnLysGlyAlaMetThrGluAsnAla    65707580    AspGluAsnAlaLeuGlnSerAspAlaLysGlyLysLeuAspSerVal    859095    AlaThrAspTyrGlyAlaAlaIleAspGlyPheIleGlyAspValSer    100105110    GlyLeuAlaAsnGlyAsnGlyAlaThrGlyAspPheAlaGlySerAsn    115120125    SerGlnMetAlaGlnValGlyAspGlyAspAsnSerProLeuMetAsn    130135140    AsnPheArgGlnTyrLeuProSerLeuProGlnSerValGluCysArg    145150155160    ProPheValPheSerAlaGlyLysProTyrGluPheSerIleAspCys    165170175    AspLysIleAsnLeuPheArgGlyValPheAlaPheLeuLeuTyrVal    180185190    AlaThrPheMetTyrValPheSerThrPheAlaAsnIleLeuArgAsn    195200205    LysGluSer    210    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 48 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    GAGACGACTAGTGGTGGCGGTGGCTCTCCATTCGTTTGTGAATATCAA48    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    TTACTAGCTAGCATAATAACGGAATACCCAAAAGAACTGG40    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    TATGCTAGCTAGTAACACGACAGGTTTCCCGACTGG36    (2) INFORMATION FOR SEQ ID NO:38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    ACCGAGCTCGAATTCGTAATCATGGTC27    (2) INFORMATION FOR SEQ ID NO:39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    AGCTGTTGAATTCGTGAAATTGTTATCCGCT31    (2) INFORMATION FOR SEQ ID NO:40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 708 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    GAGACGACTAGTGGTGGCGGTGGCTCTCCATTCGTTTGTGAATATCAAGGCCAAGGCCAA60    TCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCGGCGGCTCTGGTGGTGGTTCTGGT120    GGCGGCTCTGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGA180    GGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGATGGCAAACGCT240    AATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACAGTCTGACGCTAAAGGC300    AAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTGGTGACGTT360    TCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATG420    GCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTTACCT480    TCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTAGCGCTGGTAAACCATATGAA540    TTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATAT600    GTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGGAGTCT660    TAATCATGCCAGTTCTTTTGGGTATTCCGTTATTATGCTAGCTAGTAA708    (2) INFORMATION FOR SEQ ID NO:41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 201 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    TATGCTAGCTAGTAACACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCA60    ATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCT120    CGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCAT180    GATTACGAATTCGAGCTCGGT201    (2) INFORMATION FOR SEQ ID NO:42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    CAGGTGCAGCTCGAGCAGTCTGGG24    (2) INFORMATION FOR SEQ ID NO:43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    GAGGTGCAGCTCGAGGAGTCTGGG24    (2) INFORMATION FOR SEQ ID NO:44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    GCATGTACTAGTTTTGTCACAAGATTTGGG30    (2) INFORMATION FOR SEQ ID NO:45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    GACATCGAGCTCACCCAGTCTCCA24    (2) INFORMATION FOR SEQ ID NO:46:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:    GAAATTGAGCTCACGCAGTCTCCA24    (2) INFORMATION FOR SEQ ID NO:47:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 53 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:    GCGCCGTCTAGAACTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCAAG53    (2) INFORMATION FOR SEQ ID NO:48:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: circular    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:    SerIleSerGlyProGlyArgAlaPheTyrThrGly    1510    (2) INFORMATION FOR SEQ ID NO:49:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:    GTCGTTGACCAGGCAGCCCAG21    (2) INFORMATION FOR SEQ ID NO:50:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:    ATAGAAGTTGTTCAGCAGGCA21    (2) INFORMATION FOR SEQ ID NO:51:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:    ATTAACCCTCACTAAAG17    (2) INFORMATION FOR SEQ ID NO:52:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:    GAATTCTAAACTAGCTAGTTCG22    (2) INFORMATION FOR SEQ ID NO:53:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:    LeuGluGluSerGlyThrGluPheLysProProGlySerSerValLys    151015    ValSerCysLysAlaSerGlyGlyThrPheGlyAspTyrAlaSerAsn    202530    TyrAlaIleSerTrpValArgGlnAlaProGlyGlnGlyLeuGluTyr    354045    IleGlyGlyIleThrProThrSerGlySerAlaAspTyrAlaGlnLys    505560    PheGlnGlyArgValThrIleSerAlaAspArgPheThrProIleLeu    65707580    TyrMetGluLeuArgSerLeuArgIleGluAspThrAlaIleTyrTyr    859095    CysAlaArgGluArgArgGluArgGlyTrpAsnProArgAlaLeuArg    100105110    GlyAlaLeuAspPheTrpGlyGlnGlyThrArgValPheValSerPro    115120125    (2) INFORMATION FOR SEQ ID NO:54:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 124 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:    LeuGluGluSerGlyAlaAlaValGlnLysProGlySerSerValArg    151015    ValSerCysGlnAlaSerGlyGlyThrPheAspAsnPheAlaSerAsn    202530    TyrAlaValSerTrpValArgGlnAlaProGlyGlnGlyLeuGluTrp    354045    MetGlyGlyIleThrProThrSerGlyThrAlaThrTyrSerGlnLys    505560    PheGlnGlyArgValThrIleSerAlaAlaProLeuThrProIleIle    65707580    TyrMetGluLeuArgSerLeuArgAspAspAspThrAlaValTyrTyr    859095    CysAlaArgGluArgArgGluArgGlyTrpAsnProArgAlaLeuVal    100105110    GlyAlaLeuAspValTrpGlyGlnGlyThrThrVal    115120    (2) INFORMATION FOR SEQ ID NO:55:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:    LeuGluGluSerGlyThrGluPheLysProProGlySerSerValLys    151015    ValSerCysLysAlaSerGlyGlyThrPheGlyAspTyrAlaSerAsn    202530    TyrAlaIleSerTrpValArgGlnAlaProGlyGlnGlyLeuGluTyr    354045    IleGlyGlyIleThrProThrSerGlySerAlaAspTyrAlaGlnLys    505560    PheGlnGlyArgValThrIleSerAlaAspArgPheThrProIleLeu    65707580    TyrMetGluLeuArgSerLeuArgIleGluAspThrAlaIleTyrTyr    859095    CysAlaArgGluArgArgGluArgGlyTrpAsnProArgAlaLeuArg    100105110    GlyAlaLeuAspPheTrpGlyGlnGlyThrArgValPheValSerPro    115120125    (2) INFORMATION FOR SEQ ID NO:56:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:    LeuGluGluSerGlyAlaGluValLysLysProGlySerSerValLys    151015    ValSerCysLysAlaSerGlyGlyIlePheSerAspPheAlaSerAsn    202530    TyrAlaIleSerTrpValArgGlnAlaProGlyGlnGlyLeuGluTyr    354045    MetGlyGlyIleThrProThrSerGlySerAlaAspTyrAlaGlnLys    505560    PheGlnGlyArgValThrIleSerAlaAspAlaAlaThrProArgVal    65707580    TyrMetGluLeuArgIleLeuArgSerGluAspThrAlaValTyrPhe    859095    CysAlaArgGluArgArgGluArgGlyTrpAsnProArgAlaLeuArg    100105110    GlyAlaLeuGluValTrpGlyGlnGlyThrThrValIleValSerPro    115120125    (2) INFORMATION FOR SEQ ID NO:57:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:    LeuGluGluSerGlyAlaAlaValGlnLysProGlySerSerValArg    151015    ValSerCysGlnAlaSerGlyGlyThrPheAspAsnPheAlaSerAsn    202530    TyrAlaValSerTrpValArgGlnAlaProGlyGlnGlyLeuGluTrp    354045    MetGlyGlyIleThrProThrSerGlyThrAlaThrTyrSerGlnLys    505560    PheGlnGlyArgValThrIleSerAlaAlaProLeuThrProIleIle    65707580    TyrMetGluLeuArgSerLeuArgAspAspAspThrAlaValTyrTyr    859095    CysAlaArgGluArgArgGluArgGlyTrpAsnProArgAlaLeuVal    100105110    GlyAlaLeuAspValTrpGlyGlnGlyThrThrValIleValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:58:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:    LeuGluGlnSerGlyAlaGluValLysLysProGlySerSerValLys    151015    ValSerCysLysThrSerGlyGlyThrPheSerAspTyrAlaSerAsn    202530    HisAlaIleSerTrpValArgGlnAlaProGlyGlnGlyLeuGluTyr    354045    MetGlyGlyIleThrProThrSerGlyThrAlaAspTyrAlaGlnLys    505560    PheGlnAlaArgValThrIleSerAlaHisGluPheThrProIleVal    65707580    TyrMetGluLeuArgSerLeuArgSerAspGlnHisAlaThrTyrTyr    859095    CysAlaThrGluArgArgGluArgGlyTrpAsnProArgAlaLeuArg    100105110    GlyAlaLeuAspIleTrpGlyGlnGlyThrThrValIleValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:59:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:    LeuGluGluSerGlyGlyArgLeuValLysProGlyGlySerLeuArg    151015    LeuSerCysGluGlySerGlyPheThrPheThrAsnAlaTrpMetThr    202530    TrpValArgGlnSerProGlyLysGlyLeuGluTrpValAlaSerIle    354045    LysSerLysPheAspGlyGlySerProHisTyrAlaAlaProValGlu    505560    GlyArgPheSerIleSerArgAsnAspLeuGluAspLysMetPheLeu    65707580    GluMetSerGlyLeuLysAlaGluAspThrGlyValTyrTyrCysAla    859095    ThrLysTyrProArgTyrSerAspMetValThrGlyValArgAsnHis    100105110    PheTyrMetAspValTrpGlyLysGlyThrThrValIleValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:60:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:    LeuGluGlnSerGlyGlyGlyLeuValLysProGlyGlySerLeuArg    151015    LeuSerCysGluGlySerGlyPheThrPheThrAsnAlaTrpMetThr    202530    TrpValArgGlnSerProGlyLysGlyLeuGluTrpValAlaSerIle    354045    LysSerLysPheAspGlyGlySerProHisTyrAlaAlaProValGlu    505560    GlyArgPheThrIleSerArgAsnAspLeuGluAspLysLeuPheLeu    65707580    GluMetSerGlyLeuLysAlaGluAspThrGlyValTyrTyrCysAla    859095    ThrLysTyrProArgTyrPheAspMetMetAlaGlyValArgAsnHis    100105110    PheTyrMetAspValTrpGlyThrGlyThrThrValIleValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:61:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:    LeuGluGluSerGlyGlyGlyLeuValLysProGlyGlySerLeuArg    151015    LeuSerCysGluGlySerGlyPheThrPheThrAsnAlaTrpMetThr    202530    TrpValArgGlnSerProGlyLysGlyLeuGluTrpValAlaSerIle    354045    LysSerLysPheAspGlyGlySerProHisTyrAlaAlaProValGlu    505560    GlyArgPheThrIleSerArgAsnAspLeuGluAspLysLeuPheLeu    65707580    GluMetSerGlyLeuLysAlaGluAspThrGlyValTyrTyrCysAla    859095    ThrLysTyrProArgTyrSerAspMetMetAlaGlyValArgAsnHis    100105110    LeuTyrMetAspValTrpGlyLysGlyThrThrValIleValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:62:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:    LeuGluGluSerGlyGlyArgLeuValLysProGlyGlySerLeuArg    151015    LeuSerCysGluAlaSerGlyPheThrPheThrAsnSerTrpMetThr    202530    TrpValArgGlnSerProGlyLysGlyLeuGluTrpValAlaSerIle    354045    LysArgLysPheAspGlyGlySerProHisTyrAlaAlaProValGlu    505560    GlyArgPheSerIleSerArgAsnAspLeuGluAspLysMetPheLeu    65707580    GluMetSerGlyLeuLysAlaGluAspThrGlyValTyrTyrCysAla    859095    ThrLysTyrProArgTyrSerAspMetMetThrGlyValArgAsnHis    100105110    PheTyrMetAspValTrpGlyLysGlyThrThrValIleValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:63:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:    LeuGluGluSerGlyGlyGlyLeuValLysProGlyGlySerLeuArg    151015    LeuSerCysGluSerSerGlyPheThrPheThrAsnAlaTrpMetThr    202530    TrpValArgGlnSerProGlyLysGlyLeuGluTrpValAlaSerIle    354045    LysSerLysPheAspGlyGlySerProHisTyrAlaAlaProValGlu    505560    GlyArgPheThrIleSerArgAsnAspLeuGluAspLysLeuPheLeu    65707580    GluMetSerGlyLeuLysAlaGluAspThrGlyValTyrTyrCysAla    859095    ThrLysTyrProArgTyrSerAspMetMetAlaGlyValArgAsnHis    100105110    PheTyrMetAspValTrpGlyLysGlyThrThrValIleValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:64:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:    LeuGluGluSerGlyGlyArgLeuValLysProGlyGlySerLeuArg    151015    LeuSerCysGluGlySerGlyPheThrPheThrAsnAlaTrpMetThr    202530    TrpValArgGlnSerProGlyLysGlyLeuGluTrpValAlaSerIle    354045    LysSerLysPheAspGlyGlySerProHisTyrAlaAlaProValGlu    505560    GlyArgPheSerIleSerArgAsnAspLeuGluAspLysMetPheLeu    65707580    GluMetSerGlyLeuLysAlaGluAspThrGlyValTyrTyrCysAla    859095    ThrLysTyrProArgTyrSerAspMetMetThrGlyValArgAsnHis    100105110    PheTyrMetAspValTrpGlyLysGlyThrThrValIleValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:65:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:    LeuGluGluSerGlyGlyGlyLeuValLysProGlyGlySerLeuArg    151015    LeuSerCysAlaGlySerGlyPheThrPheThrAsnAlaTrpMetThr    202530    TrpValArgGlnSerProGlyLysGlyLeuGluTrpValAlaSerIle    354045    LysSerLysPheAspGlyGlySerSerHisTyrProGlyProValGlu    505560    GlyArgPheThrIleSerArgAsnTyrIleGluAspLysLeuPheLeu    65707580    GluMetSerGlyLeuLysAlaGluAspThrGlyValTyrTyrCysAla    859095    ThrLysTyrProArgTyrTyrAspMetMetArgGlyValArgAsnHis    100105110    TyrTyrMetAspValTrpGlyLysGlyThrThrValIleValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:66:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 124 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:    LeuGluGlnSerGlyAlaGluValLysLysProGlyAlaSerValLys    151015    ValSerCysGlnAlaSerGlyTyrArgPheSerAsnPheValIleHis    202530    TrpValArgGlnAlaProGlyGlnArgPheGluTrpMetGlyTrpIle    354045    AsnProTyrAsnGlyAsnLysGluPheSerAlaLysPheGlnAspArg    505560    ValThrPheThrAlaAspThrSerAlaAsnThrAlaTyrMetGluLeu    65707580    ArgSerLeuArgSerAlaAspThrAlaValTyrTyrCysAlaArgVal    859095    GlyProTyrSerTrpAspAspSerProGlnAspAsnTyrTyrMetAsp    100105110    ValTrpGlyLysGlyThrThrValIleValSerSer    115120    (2) INFORMATION FOR SEQ ID NO:67:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 124 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:    LeuGluGlnSerGlyAlaGluValLysLysProGlyAlaSerValLys    151015    ValSerCysGlnAlaSerGlyTyrArgPheSerAsnPheValIleHis    202530    TrpValArgGlnAlaProGlyGlnArgPheGluTrpMetGlyTrpIle    354045    AsnProTyrAsnGlyAsnLysGluPheSerAlaLysPheGlnAspArg    505560    ValThrPheThrAlaAspThrAspAlaAsnThrAlaTyrMetGluLeu    65707580    ArgSerLeuArgSerAlaAspThrAlaIleTyrTyrCysAlaArgVal    859095    GlyProTyrThrTrpAspAspSerProGlnAspAsnTyrTyrMetAsp    100105110    ValTrpGlyLysGlyThrLysValIleValSerSer    115120    (2) INFORMATION FOR SEQ ID NO:68:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 124 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:    LeuGluGlnSerGlyAlaGluValLysLysProGlyAlaSerValLys    151015    ValSerCysGlnAlaSerGlyTyrArgPheSerAsnPheValIleHis    202530    TrpValArgGlnAlaProGlyGlnArgPheGluTrpMetGlyTrpIle    354045    AsnProTyrAsnGlyAsnLysGluPheSerAlaLysPheGlnAspArg    505560    ValThrPheThrAlaAspThrAspAlaAsnThrAlaTyrMetGluLeu    65707580    ArgSerLeuArgSerThrAspThrAlaIleTyrTyrCysAlaArgVal    859095    GlyProTyrThrTrpAspAspSerProGlnAspAsnTyrTyrMetAsp    100105110    ValTrpGlyLysGlyThrLysValIleValSerSer    115120    (2) INFORMATION FOR SEQ ID NO:69:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 130 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:    LeuGluGluSerGlyGlyGlyLeuValLysProGlyGlySerLeuArg    151015    LeuSerCysValGlySerGlyPheThrPheSerSerAlaTrpMetAla    202530    TrpValArgGlnAlaProGlyArgGlyLeuGluTrpValGlyLeuIle    354045    LysSerLysAlaAspGlyGluThrThrAspTyrAlaThrProValLys    505560    GlyArgPheSerIleSerArgAsnAsnLeuGluAspThrValTyrLeu    65707580    GlnMetAspSerLeuArgAlaAspAspThrAlaValTyrTyrCysAla    859095    ThrGlnLysProArgTyrPheAspLeuLeuSerGlyGlnTyrArgArg    100105110    ValAlaGlyAlaPheAspValTrpGlyHisGlyThrThrValThrVal    115120125    SerPro    130    (2) INFORMATION FOR SEQ ID NO:70:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 130 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:    LeuGluGluSerGlyGlyGlyLeuValLysAlaGlyGlySerLeuArg    151015    LeuSerCysValGlySerGlyPheThrPheSerSerAlaTrpMetAla    202530    TrpValGlyGlnAlaProGlyArgGlyLeuGluTrpValGlyLeuIle    354045    LysSerLysAlaAspGlyGluThrThrAspTyrAlaThrProValLys    505560    GlyArgPheSerIleSerArgAsnAsnLeuGluAspThrValTyrLeu    65707580    GlnMetAspSerLeuArgAlaAspAspThrAlaValTyrTyrCysAla    859095    ThrGlnLysProArgTyrPheAspLeuLeuSerGlyGlnTyrArgArg    100105110    ValAlaGlyAlaPheAspValTrpGlyHisGlyThrThrValThrVal    115120125    SerPro    130    (2) INFORMATION FOR SEQ ID NO:71:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 130 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:    LeuGluGluSerGlyGlyGlyLeuIleLysProGlyGlySerLeuArg    151015    LeuSerCysValGlySerGlyPheThrPheSerSerAlaTrpMetThr    202530    TrpValArgGlnAlaProGlyLysGlyLeuGluTrpIleGlyLeuIle    354045    LysSerLysAlaAspGlyGluThrThrAspTyrAlaThrProValLys    505560    GlyArgPheThrIleSerArgAsnAsnLeuGluAsnThrValTyrLeu    65707580    GlnMetAspSerLeuArgAlaAspAspThrAlaValTyrTyrCysAla    859095    ThrGlnLysProSerTyrTyrAsnLeuLeuSerGlyGlnTyrArgArg    100105110    ValAlaGlyAlaPheAspValTrpGlyHisGlyThrThrValThrVal    115120125    SerPro    (2) INFORMATION FOR SEQ ID NO:72:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:    LeuGluGluSerGlyGluAlaValValGlnProGlyArgSerLeuArg    151015    LeuSerCysAlaAlaSerGlyPheIlePheArgAsnTyrAlaMetHis    202530    TrpValArgGlnAlaProGlyLysGlyLeuGluTrpValAlaLeuIle    354045    LysTyrAspGlyArgAsnLysTyrTyrAlaAspSerValLysGlyArg    505560    PheThrIleSerArgAspAsnSerLysAsnThrLeuTyrLeuGlnMet    65707580    AsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaArgAsp    859095    IleGlyLeuLysGlyGluHisTyrAspIleLeuThrAlaTyrGlyPro    100105110    AspTyrTrpGlyGlnGlyThrLeuValThrValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:73:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:    LeuGluGlnSerGlyGluAlaValValGlnProGlyThrSerLeuArg    151015    LeuSerCysAlaAlaSerGlyPheThrPheArgAsnTyrAlaMetHis    202530    TrpValArgGlnAlaProGlyLysGlyLeuGluTrpValAlaLeuIle    354045    LysTyrAspGlyArgAsnLysTyrTyrAlaAspSerValLysGlyArg    505560    PheSerIleSerArgAspAsnSerLysAsnThrLeuTyrLeuGluMet    65707580    AsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaArgAsp    859095    IleGlyLeuLysGlyGluHisTyrAspIleLeuThrAlaTyrGlyPro    100105110    AspTyrTrpGlyGlnGlyAlaLeuValThrValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:74:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:    LeuGluGlnSerGlyGluAlaValValGlnProGlyArgSerLeuArg    151015    LeuSerCysAlaAlaSerGlyPheIlePheArgAsnTyrAlaMetHis    202530    TrpValArgGlnAlaProGlyLysGlyLeuGluTrpValAlaLeuIle    354045    LysTyrAspGlyArgAsnLysTyrTyrAlaAspSerValLysGlyArg    505560    PheThrIleSerArgAspAsnSerLysAsnThrLeuTyrLeuGlnMet    65707580    AsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaArgAsp    859095    IleGlyLeuLysGlyGluHisTyrAspIleLeuThrAlaTyrGlyPro    100105110    AspTyrTrpGlyGlnGlyThrLeuValThrValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:75:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:    LeuGluGluSerGlyGluAlaValValGlnProGlyThrSerLeuArg    151015    LeuSerCysAlaAlaSerGlyPheThrPheArgAsnTyrAlaMetHis    202530    TrpValArgGlnAlaProGlyLysGlyLeuGluTrpValAlaLeuIle    354045    LysTyrAspGlyArgAsnLysTyrTyrAlaAspSerValLysGlyArg    505560    PheSerIleSerArgAspAsnSerLysAsnThrLeuTyrLeuGluMet    65707580    AsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaArgAsp    859095    IleGlyLeuLysGlyGluHisTyrAspIleLeuThrAlaTyrGlyPro    100105110    AspTyrTrpGlyGlnGlyAlaLeuValThrValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:76:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:    LeuGluGlnSerGlyGluAlaValValGlnProGlyArgSerLeuArg    151015    LeuSerCysAlaAlaSerGlyPheThrPheArgAsnTyrAlaMetHis    202530    TrpValArgGlnAlaProGlyLysGlyLeuGluTrpValAlaLeuIle    354045    LysTyrAspGlyArgAsnLysTyrTyrAlaAspSerValLysGlyArg    505560    PheThrIleSerArgAspAsnSerLysAsnThrLeuTyrLeuGlnMet    65707580    AsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaArgAsp    859095    IleGlyLeuLysAlaGluHisTyrAspIleLeuThrAlaTyrGlyPro    100105110    AspTyrTrpGlyGlnGlyThrLeuValThrValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:77:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:    LeuGluGlnSerGlyGluAlaValValGlnProGlyArgSerLeuArg    151015    LeuSerCysAlaAlaSerGlyPheIlePheArgAsnTyrAlaMetHis    202530    TrpValArgGlnAlaProGlyLysGlyLeuGluTrpValAlaLeuIle    354045    LysTyrAspGlyArgAsnLysTyrTyrAlaAspSerValLysGlyArg    505560    PheThrIleSerArgAspAsnSerLysAsnThrLeuTyrLeuGlnMet    65707580    AsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaArgAsp    859095    IleGlyLeuLysGlyGluHisTyrAspIleLeuThrAlaTyrGlyPro    100105110    AspTyrTrpGlyGlnGlyThrLeuValThrValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:78:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:    LeuGluGlnSerGlyGlyGlyValValLysProGlyGlySerLeuArg    151015    LeuSerCysGluGlySerGlyPheThrPheProAsnAlaTrpMetThr    202530    TrpValArgGlnSerProGlyLysGlyLeuGluTrpValAlaSerIle    354045    LysSerLysPheAspGlyGlySerProHisTyrAlaAlaProValGlu    505560    GlyArgPheThrIleSerArgAsnAspLeuGluAspLysValPheLeu    65707580    GlnMetAsnGlyLeuLysAlaGluAspThrGlyValTyrTyrCysAla    859095    ThrArgTyrProArgTyrSerGluMetMetGlyGlyValArgLysHis    100105110    PheTyrMetAspValTrpGlyLysGlyThrThrValSerValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:79:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:    LeuGluGluSerGlyGlyGlyValValLysProGlyGlySerLeuArg    151015    LeuSerCysGluGlySerGlyPheThrPheProAsnAlaTrpMetThr    202530    TrpValArgGlnSerProGlyLysGlyLeuGluTrpValAlaSerIle    354045    LysSerLysPheAspGlyGlySerProHisTyrAlaAlaProValGlu    505560    GlyArgPheThrIleSerArgAsnAspLeuGluAspLysValPheLeu    65707580    GlnMetAsnGlyLeuLysAlaGluAspThrGlyValTyrTyrCysAla    859095    ThrArgTyrProArgTyrSerGluMetMetGlyGlyValArgLysHis    100105110    PheTyrMetAspValTrpGlyLysGlyThrThrValSerValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:80:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 122 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:    LeuGluGluSerGlyGlyGlyLeuValGlnProGlyArgSerLeuArg    151015    ValSerCysGluAlaSerGlyPheThrPheSerSerTyrGluMetAsn    202530    TrpValArgGlnAlaProGlyLysGlyLeuGluTrpValSerGlnIle    354045    SerSerSerGlySerArgThrTyrTyrAlaAspSerValLysGlyArg    505560    PheThrIleSerArgAspAsnAlaLysAsnSerLeuTyrLeuGluMet    65707580    ThrSerLeuArgValAspAspThrAlaValTyrTyrCysAlaArgGly    859095    ArgArgLeuValThrPheGlyGlyValValSerGlyGlyAsnIleTrp    100105110    GlyGlnGlyThrMetValThrValSerSer    115120    (2) INFORMATION FOR SEQ ID NO:81:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 126 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:    LeuGluGlnSerGlyGlyGlyValValGlnProGlyArgSerLeuArg    151015    LeuSerCysAlaGlySerGlyPheAsnPheSerAspAspThrMetHis    202530    TrpValArgGlnAlaProGlyLysGlyLeuGluTrpValAlaValIle    354045    SerTyrGluGlySerAspLysTyrTyrAlaAspSerValLysGlyArg    505560    PheThrIleSerArgAspAsnSerGluAsnThrLeuTyrLeuGlnMet    65707580    AspSerLeuArgAlaAspAspThrAlaLeuTyrTyrCysAlaArgAsn    859095    ThrArgGluAsnIleGluAlaAspGlyThrAlaTyrTyrSerTyrTyr    100105110    MetAspValTrpGlyLysGlyThrThrValThrValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:82:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:    GluLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArg    151015    ValThrIleThrCysArgAlaSerGlnGlyIleSerAsnTyrLeuAla    202530    TrpTyrGlnGlnLysProGlyLysValProArgLeuLeuIleTyrAla    354045    AlaSerThrLeuGlnProGlyValProSerArgPheSerGlySerGly    505560    SerGlyThrAspPheThrLeuThrIleSerSerLeuGlnProGluAsp    65707580    ValAlaThrTyrTyrCysGlnLysTyrAsnSerAlaProArgThrPhe    859095    GlyGlnGlyThrLysValGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:83:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 106 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:    GluLeuThrGlnSerProSerSerLeuSerAlaSerIleGlyAspArg    151015    ValThrIleThrCysArgAlaSerGlnGlyIleAsnAsnTyrLeuAla    202530    TrpTyrGlnGlnArgProGlyLysValProArgLeuLeuIleTyrAla    354045    AlaSerThrLeuGlnSerGlyValProThrArgPheSerGlySerGly    505560    SerGlyThrAspPheThrLeuThrIleSerSerLeuGlnProGluAsp    65707580    ValAlaThrTyrTyrCysGlnLysTyrAsnSerValProArgThrPhe    859095    GlyGlyGlyThrLysValGluIleLysArg    100105    (2) INFORMATION FOR SEQ ID NO:84:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:    GluLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArg    151015    ValThrIleThrCysArgAlaSerGlnGlyIleSerAsnTyrLeuAla    202530    TrpTyrGlnGlnLysProGlyLysValProLysLeuLeuIleTyrAla    354045    AlaSerThrLeuGlnSerGlyValProSerArgPheSerGlySerGly    505560    SerGlyThrAspPheThrLeuThrIleSerSerLeuGlnProGluAsp    65707580    ValAlaThrTyrTyrCysGlnLysTyrAsnSerAlaProArgThrPhe    859095    GlyGlnGlyThrLysValGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:85:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 106 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:    GluLeuThrGlnSerProSerSerLeuSerAlaSerIleGlyAspArg    151015    ValThrIleThrCysArgAlaSerGlnGlyIleAsnAsnTyrLeuAla    202530    TrpTyrGlnGlnArgProGlyLysAlaProAsnLeuLeuIleTyrAla    354045    AlaSerThrLeuGlnSerGlyValProProArgPheSerGlySerGly    505560    SerGlyThrAspPheThrLeuThrIleSerSerLeuGlnProGluAsp    65707580    ValAlaThrTyrTyrCysGlnLysTyrAsnSerValProHisThrPhe    859095    GlyGlyGlyThrLysValGluIleLysArg    100105    (2) INFORMATION FOR SEQ ID NO:86:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 108 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:    GluLeuThrGlnSerProGlyThrLeuSerLeuSerProGlyGluArg    151015    AlaThrLeuSerCysArgAlaSerGlnSerValIleSerAsnTyrLeu    202530    AlaTrpTyrGlnGlnLysProGlyGlnAlaProArgLeuLeuIleTyr    354045    GlyValSerAsnArgAlaThrGlyIleProAspArgPheSerGlySer    505560    GlySerGlyThrAspPheThrLeuThrIleSerArgLeuGluProGlu    65707580    AspPheAlaValTyrSerCysGlnGlnTyrGlyThrSerProTrpThr    859095    PheGlyGlnGlyThrLysValGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:87:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:    GluLeuThrGlnSerProGlyThrLeuSerLeuSerProGlyGluArg    151015    AlaThrLeuSerCysArgAlaSerGlnSerValSerAsnAsnTyrLeu    202530    AlaTrpTyrGlnGlnArgProGlyGlnAlaProArgLeuLeuIleTyr    354045    GlyAlaSerAsnArgAlaThrGlyIleProAspArgPheSerGlySer    505560    GlySerGlyThrAlaPheThrLeuThrIleSerSerLeuGlnProGlu    65707580    AspValAlaIleTyrTyrCysGlnGlnTyrHisSerSerProTyrThr    859095    PheGlyGlnGlyThrLysLeuGluIleLysArg    100105    (2) INFORMATION FOR SEQ ID NO:88:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 108 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:    GluLeuThrGlnSerProGlyThrLeuSerLeuSerProGlyGluArg    151015    AlaThrLeuSerCysArgAlaSerHisArgValAsnAsnAsnPheLeu    202530    AlaTrpTyrGlnGlnLysProGlnAlaProArgLeuLeuIleSerGly    354045    AlaSerThrArgAlaThrGlyIleProAspArgPheSerGlySerGly    505560    SerGlyThrAspPheThrLeuThrIleSerArgLeuGluProAspAsp    65707580    PheAlaValTyrTyrCysGlnGlnTyrGlyAspSerProLeuTyrSer    859095    PheGlyGlnGlyThrLysLeuGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:89:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 105 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:    GluLeuThrGlnSerProAlaSerValSerAlaSerValGlyAspThr    151015    ValThrIleThrCysArgAlaSerGlnAspIleHisAsnTrpLeuAla    202530    TrpTyrGlnGlnGlnProGlyLysAlaProLysLeuLeuIleTyrAla    354045    AlaSerSerLeuGlnSerGlyValProSerArgPheSerGlyArgGly    505560    SerGlyThrAspPheThrLeuThrIleSerSerLeuGlnProGluAsp    65707580    PheAlaThrTyrTyrCysGlnGlnGlyAsnSerPheProLysPheGly    859095    ProGlyThrValValAspIleLysArg    100105    (2) INFORMATION FOR SEQ ID NO:90:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:    GluLeuThrGlnSerProGlyThrLeuSerLeuSerProGlyGluArg    151015    AlaThrLeuSerCysArgAlaSerGlnSerLeuSerAsnAsnTyrLeu    202530    AlaTrpTyrGlnGlnLysProGlyGlnAlaProArgLeuLeuIleTyr    354045    GlySerSerThrArgGlyThrGlyIleProAspArgPheSerGlyGly    505560    GlySerGlyThrAspPheThrLeuThrIleSerArgLeuGluProGlu    65707580    AspPheAlaValTyrTyrCysGlnHisTyrGlyAsnSerValTyrThr    859095    PheGlyGlnGlyThrLysLeuGluIleLysArg    100105    (2) INFORMATION FOR SEQ ID NO:91:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 104 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:    GlnSerProAspThrLeuSerLeuAsnProGlyGluArgAlaThrLeu    151015    SerCysArgAlaSerHisArgIleSerSerLysArgLeuAlaTrpTyr    202530    GlnHisLysArgGlyGlnAlaProArgLeuLeuIleTyrValCysPro    354045    AsnArgAlaGlyGlyValProAspArgPheSerGlySerGlySerGly    505560    ThrAspPheThrLeuThrTyrSerArgLeuGluProGluAspPheAla    65707580    MetTyrTyrCysGlnTyrTyrGlyGlySerSerTyrThrPheGlyGln    859095    GlyThrLysValGluIleThrArg    100    (2) INFORMATION FOR SEQ ID NO:92:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 104 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:    GlnSerProSerHisLeuSerLeuSerProGlyGluArgAlaIleLeu    151015    SerCysArgAlaSerGlnArgValSerAlaProTyrLeuAlaTrpTyr    202530    GlnGlnArgProGlyGlnAlaProArgLeuValIleTyrGlyAlaSer    354045    ThrArgAlaThrAspIleProAspArgPheSerGlySerGlySerGly    505560    ThrAspPheThrLeuThrIleSerArgLeuGluProGluAspPheAla    65707580    IleTyrTyrCysGlnValTyrGlyGlnSerProValLeuPheGlyGln    859095    GlyThrLysLeuGluMetLysArg    100    (2) INFORMATION FOR SEQ ID NO:93:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 105 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:    GlnSerProGlyThrLeuSerLeuSerProGlyAspArgAlaThrLeu    151015    SerCysArgAlaSerGlnSerLeuSerSerSerPheLeuAlaTrpTyr    202530    GlnGlnLysProGlyGlnAlaProArgLeuLeuIleTyrSerAlaSer    354045    MetArgAlaThrGlyIleProAspArgPheArgGlySerValSerGly    505560    ThrAspPheThrLeuThrIleThrArgLeuGluProGluAspPheAla    65707580    ValTyrTyrCysGlnArgPheGlyThrSerProLeuTyrThrPheGly    859095    GlnGlyThrLysLeuGluMetLysArg    100105    (2) INFORMATION FOR SEQ ID NO:94:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 104 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:    GlnSerProGlyThrLeuSerLeuSerProGlyGluArgAlaThrLeu    151015    SerCysArgAlaSerGlnSerPheSerSerAsnPheLeuAlaTrpTyr    202530    GlnGlnLysProGlyGlnAlaProArgLeuLeuIleTyrValHisPro    354045    AsnArgAlaThrGlyValProAspArgPheSerGlySerGlySerGly    505560    ThrAspPheThrLeuThrIleArgArgLeuGluProGluAspPheAla    65707580    ValTyrTyrCysGlnGlnTyrGlyAlaSerLeuValSerPheGlyPro    859095    GlyThrLysValHisIleLysArg    100    (2) INFORMATION FOR SEQ ID NO:95:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 108 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:    GluLeuThrGlnSerProGlyThrLeuSerLeuSerProGlyGluArg    151015    AlaThrPheSerCysArgSerSerHisSerIleArgSerArgArgVal    202530    AlaTrpTyrGlnHisLysProGlyGlnAlaProArgLeuValIleHis    354045    GlyValSerAsnArgAlaSerGlyIleSerAspArgPheSerGlySer    505560    GlySerGlyThrAspPheThrLeuThrIleThrArgValGluProGlu    65707580    AspPheAlaLeuTyrTyrCysGlnValTyrGlyAlaSerSerTyrThr    859095    PheGlyGlnGlyThrLysLeuGluArgLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:96:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 108 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:    GluLeuThrGlnSerProGlyThrLeuSerLeuThrProGlyGluArg    151015    AlaThrLeuSerCysArgThrSerHisSerIleArgSerArgArgLeu    202530    AlaTrpTyrGlnValLysGlyGlyGlnAlaProArgLeuLeuIleTyr    354045    GlyValSerAsnArgAlaGlyGlyIleProAspArgPheSerGlySer    505560    GlySerGlyThrAspPheThrLeuThrIleSerArgLeuGluProGlu    65707580    AspPheAlaValTyrTyrCysGlnGlnTyrGlySerSerArgTyrThr    859095    PheGlyGlnGlyThrLysLeuGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:97:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:    GluLeuThrGlnAlaProGlyThrLeuSerLeuSerProGlyGluArg    151015    AlaThrPheSerCysArgSerSerHisSerIleArgSerArgArgVal    202530    ArgTrpTyrGlnHisLysProGlyGlnAlaProArgLeuValIleHis    354045    GlyValSerAsnArgAlaSerGlyIleSerAspArgPheSerGlySer    505560    GlySerGlyThrAspPheThrLeuThrIleThrArgValGluProGlu    65707580    AspPheAlaLeuTyrTyrCysGlnValTyrGlyAlaSerSerTyrThr    859095    PheGlyGlnGlyThrLysLeuGluArgLysArg    100105    (2) INFORMATION FOR SEQ ID NO:98:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 108 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:    GluLeuThrGlnAlaProGlyThrLeuSerLeuSerProGlyAspArg    151015    AlaThrPheSerCysArgSerSerHisAsnIleArgSerArgArgVal    202530    AlaTrpTyrGlnHisLysProGlyGlnAlaProArgLeuValIleHis    354045    GlyValSerAsnArgAlaSerGlyIleSerAspArgPheSerGlySer    505560    GlySerGlyThrAspPheThrLeuThrIleThrArgLeuGluProGlu    65707580    AspPheAlaLeuTyrTyrCysGlnValTyrGlyAlaSerSerTyrThr    859095    PheGlyGlnGlyThrLysLeuAspPheLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:99:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 108 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:    GluLeuThrGlnSerProGlyThrLeuSerLeuSerProGlyGluArg    151015    AlaThrLeuSerCysArgAlaGlyGlnSerIleSerSerAsnTyrLeu    202530    AlaTrpTyrGlnGlnLysProGlyGlnAlaProArgLeuLeuIleTyr    354045    GlyAlaSerAsnArgAlaThrGlyIleProAspArgPheSerGlySer    505560    GlySerGlyThrAspPheThrLeuSerIleSerArgLeuGluProGlu    65707580    AspPheAlaValTyrTyrCysGlnGlnTyrGlyThrSerProTyrThr    859095    PheGlyGlnGlyThrGlnLeuAspIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:100:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 104 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:    GlnSerProGlyThrLeuSerLeuSerProGlyGluArgAlaThrLeu    151015    SerCysArgAlaSerGlnSerLeuSerAsnAsnTyrLeuAlaTrpTyr    202530    GlnGlnLysProGlyGlnAlaProArgLeuLeuIleTyrGlySerSer    354045    ThrArgAlaThrGlyIleProAspArgPheSerGlyGlyGlySerGly    505560    ThrAspPheThrLeuThrIleSerArgLeuGluProGluAspPheAla    65707580    ValTyrTyrCysGlnGlnTyrGlyAsnSerValTyrThrPheGlyGln    859095    GlyThrLysLeuGluIleLysArg    100    (2) INFORMATION FOR SEQ ID NO:101:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 106 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:    GluLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArg    151015    ValThrIleThrCysArgThrSerGlnGlyIleSerAsnTyrLeuAla    202530    TrpTyrGlnGlnLysProGlyLysValProLysLeuLeuIleTyrGly    354045    AlaSerThrLeuGlnSerGlyGlyProSerArgPheSerGlySerGly    505560    SerGlyThrAspPheThrLeuThrIleAsnSerLeuGlnProGluAsp    65707580    ValAlaThrTyrSerCysGlnAsnTyrAspSerAlaProTrpThrPhe    859095    GlyGlnGlyThrLysValAspIleLysArg    100105    (2) INFORMATION FOR SEQ ID NO:102:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 108 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:    GluLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArg    151015    ValThrIleThrCysArgAlaSerGlnSerIleSerAsnTyrLeuAsn    202530    TrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyrAla    354045    AlaSerSerLeuGlnArgGlyValProSerArgPheSerGlySerGly    505560    SerGlyThrAspPheThrLeuSerIleSerSerLeuGlnProGluAsp    65707580    PheAlaThrTyrTyrCysGlnGlnSerTyrSerIleProProLeuThr    859095    PheGlyGlyGlyThrLysValGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:103:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:    GluLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArg    151015    ValThrIleThrCysArgAlaSerGlnAsnIleAsnAsnTyrLeuAsn    202530    TrpTyrGlnGlnLysProGlyGluAlaProLysLeuLeuIleHisThr    354045    AlaPheAsnLeuGlnSerGlyValProSerArgPheSerGlyThrAla    505560    SerGlyThrGluPheThrLeuThrIleArgSerLeuGlnProGluAsp    65707580    PheAlaThrTyrTyrCysGlnGlnSerTyrSerThrProTyrThrPhe    859095    GlyGlnGlyThrLysValGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:104:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:    GluLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArg    151015    ValThrIleThrCysArgAlaSerGlnSerIleSerSerTyrLeuAsn    202530    TrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyrAla    354045    AlaSerSerLeuGlnSerGlyValProSerArgPheSerGlySerGly    505560    SerGlyThrAspPheThrLeuThrIleSerSerLeuGlnProGluAsp    65707580    PheAlaThrTyrTyrCysGlnGlnSerTyrSerThrProTyrThrPhe    859095    GlyGlnGlyThrLysLeuGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:105:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:    GluLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArg    151015    ValThrIleThrCysArgAlaSerGlnSerIleSerSerTyrLeuAsn    202530    TrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyrAla    354045    AlaSerSerLeuGlnSerGlyValProSerArgPheSerGlySerGly    505560    SerGlyThrAspPheThrLeuThrIleSerSerLeuGlnProGluAsp    65707580    PheAlaThrTyrTyrCysGlnGlnSerTyrSerThrProGlnThrPhe    859095    GlyGlnGlyThrLysLeuGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:106:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 104 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:    GlnSerProSerSerLeuSerAlaSerValGlyAspArgValThrIle    151015    ThrCysArgAlaSerGlnThrIleSerSerTyrLeuAsnTrpTyrGln    202530    GlnLysProGlyLysAlaProLysLeuLeuIleTyrAlaAlaSerSer    354045    LeuGlnSerGlyValProSerArgPheSerGlyGlyGlySerGlyThr    505560    AspPheThrLeuThrIleSerSerLeuGlnProGluAspPheAlaThr    65707580    TyrTyrCysGlnGlnSerTyrSerThrProTyrThrPheGlyGlnGly    859095    ThrLysLeuGluIleLysArgThr    100    (2) INFORMATION FOR SEQ ID NO:107:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:    GluLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArg    151015    ValThrIleThrCysGlnAlaSerGlnAspIleArgAsnTyrLeuAsn    202530    TrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyrAsp    354045    AlaSerAsnSerGluThrGlyValProSerArgPheSerGlySerGly    505560    SerGlyArgAspPheThrPheThrIleSerSerLeuGlnProGluAsp    65707580    ValAlaThrTyrTyrCysGlnGlnHisGlnAsnValProLeuThrPhe    859095    GlyGlyGlyThrLysValGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:108:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:    GluLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArg    151015    ValThrIleThrCysGlnAlaSerGlnAspIleSerAsnHisLeuAsn    202530    TrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyrAsp    354045    AlaSerAsnLeuGluThrGlyValProSerArgPheSerGlySerGly    505560    SerGlyThrAspPheThrPheThrIleSerSerLeuGlnProGluAsp    65707580    IleAlaThrTyrTyrCysGlnGlnTyrAspAsnLeuProLeuThrPhe    859095    GlyGlyGlyThrLysValGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:109:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 108 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:    GluLeuThrGlnSerProSerSerLeuSerAlaSerValGlyAspArg    151015    IleThrIleThrCysArgAlaSerGlnThrIleAsnAsnTyrLeuAsn    202530    TrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyrGly    354045    AlaSerAsnLeuGlnSerGlyValProSerArgPheSerGlySerGly    505560    SerGlyThrAspPheThrLeuThrIleSerSerLeuGlnProGluAsp    65707580    PheAlaThrTyrPheCysGlnGlnSerTyrAsnThrProProTrpThr    859095    PheGlyGlnGlyThrLysValGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:110:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 108 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:    GluLeuThrGlnSerProGlyThrLeuSerLeuSerProGlyGluArg    151015    AlaThrLeuSerCysArgAlaSerGlnArgValAsnSerAsnTyrLeu    202530    AlaTrpTyrGlnGlnLysProGlyGlnThrProArgValValIleTyr    354045    SerThrSerArgArgAlaThrGlyValProAspArgPheSerGlySer    505560    GlySerGlyThrAspPheThrLeuThrIleSerArgLeuGluProGlu    65707580    AspPheAlaValTyrTyrCysGlnGlnPheGlyAspAlaGlnTyrThr    859095    PheGlyGlnGlyThrLysLeuGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:111:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 93 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:    GluArgAlaThrLeuSerCysArgAlaSerGlnArgValAsnSerAsn    151015    TyrLeuAlaTrpTyrGlnGlnLysProGlyGlnThrProArgValVal    202530    IleTyrSerThrSerArgArgAlaThrGlyValProAspArgPheSer    354045    GlySerGlySerGlyThrAspPheThrLeuThrIleSerArgLeuGlu    505560    ProGluAspPheAlaValTyrTyrCysGlnGlnPheGlyAspAlaGln    65707580    TyrThrPheGlyGlnGlyThrLysLeuGluIleLysArg    8590    (2) INFORMATION FOR SEQ ID NO:112:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 104 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:    ThrGlnSerProSerSerValSerAlaSerValGlyAspThrValThr    151015    PheThrCysArgAlaSerGlnAspIleArgAsnTyrLeuAsnTrpTyr    202530    HisGlnLysProGlyLysAlaProLysLeuLeuIleSerAspAlaSer    354045    AspLeuGluIleGlyValProSerArgPheSerGlySerGlySerAla    505560    ThrTyrPheSerPheThrIleSerSerLeuGlnProGluAspIleGly    65707580    ThrTyrTyrCysGlnGlnTyrAlaAspLeuIleThrPheGlyGlyGly    859095    ThrLysValGluIleLysArgThr    100    (2) INFORMATION FOR SEQ ID NO:113:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 96 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:    SerProGlyGluArgAlaThrLeuSerCysArgAlaSerGlnSerVal    151015    GlyThrAsnLeuAlaTrpTyrGlnGlnLysProGlyGlnAlaProArg    202530    LeuLeuIlePheAspAlaSerThrArgAspThrTyrIleProAspThr    354045    PheSerGlySerGlySerGlyThrAspPheAlaLeuThrIleSerSer    505560    LeuGlnSerGluAspPheGlyPheTyrTyrCysGlnGlnTyrAspAsn    65707580    TrpProProThrPheGlyGlnGlyThrLysLeuGluValLysArgThr    859095    (2) INFORMATION FOR SEQ ID NO:114:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:    GluLeuThrGlnSerProGlyThrLeuSerLeuSerProGlyAspArg    151015    AlaThrPheSerCysArgSerSerHisAsnIleArgSerArgArgVal    202530    AlaTrpTyrGlnHisLysProGlyGlnAlaProArgLeuValIleHis    354045    GlyValSerAsnArgAlaSerGlyIleSerAspArgPheSerGlySer    505560    GlySerGlyThrAspPheThrLeuThrIleThrArgLeuGluProGlu    65707580    AspPheAlaLeuTyrTyrCysGlnValTyrGlyAlaSerSerTyrThr    859095    PheGlyGlnGlyThrLysLeuAspPheLysArg    100105    (2) INFORMATION FOR SEQ ID NO:115:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:    GluLeuThrGlnSerProGlyThrLeuSerLeuSerProGlyGluArg    151015    AlaThrPheSerCysArgSerSerHisAsnIleArgSerArgArgVal    202530    AlaTrpTyrGlnHisLysProGlyGlnAlaProArgLeuValIleHis    354045    GlyValSerAsnArgAlaThrGlyIleSerAspArgPheSerGlySer    505560    GlySerGlyThrAspPheThrLeuThrIleThrArgLeuGluProGlu    65707580    AspPheAlaLeuTyrTyrCysGlnValTyrGlyAlaSerSerTyrThr    859095    PheGlyGlnGlyThrLysLeuAspPheLysArg    100105    (2) INFORMATION FOR SEQ ID NO:116:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:    GluLeuThrGlnSerProAspThrLeuSerLeuAsnValGlyGluArg    151015    AlaThrLeuSerCysArgAlaSerHisArgIleSerSerArgArgLeu    202530    AlaTrpTyrGlnHisLysArgGlyGlnAlaProArgLeuLeuIleTyr    354045    GlyValSerSerArgAlaGlyGlyValProAspArgPheSerGlySer    505560    GlySerGlyThrAspPheSerLeuThrIleSerArgLeuGluProGlu    65707580    AspPheAlaMetTyrTyrCysGlnThrTyrGlyGlySerSerTyrThr    859095    PheGlyGlnGlyThrLysValAspIleLysArg    100105    (2) INFORMATION FOR SEQ ID NO:117:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:    GluLeuThrGlnSerProAspThrLeuSerLeuAsnAlaGlyGluArg    151015    AlaThrLeuSerCysArgAlaSerHisArgIleSerSerArgArgLeu    202530    AlaTrpTyrGlnHisLysArgGlyGlnAlaProArgLeuLeuIleTyr    354045    GlyValSerAsnArgAlaGlyGlyValProAspArgPheSerGlySer    505560    GlySerGlyThrAspPheSerLeuThrIleSerArgLeuGluProGlu    65707580    AspPheAlaIleTyrTyrCysGlnThrTyrGlyGlySerSerTyrThr    859095    PheGlyGlnGlyThrThrValAspIleLysArg    100105    (2) INFORMATION FOR SEQ ID NO:118:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:    GluLeuThrGlnSerProAspThrLeuSerLeuAsnThrGlyGluArg    151015    AlaThrLeuSerCysArgAlaSerHisArgIleGlySerArgArgLeu    202530    AlaTrpTyrGlnHisArgArgGlyGlnAlaProArgLeuLeuIleTyr    354045    GlyValSerAsnArgAlaGlyGlyValProAspArgPheSerGlySer    505560    GlySerGlyThrAspPheThrLeuThrIleSerArgLeuGluProGlu    65707580    AspPheAlaIleTyrTyrCysGlnThrTyrGlyGlySerSerTyrThr    859095    PheGlyGlnGlyThrLysValAspIleLysArg    100105    (2) INFORMATION FOR SEQ ID NO:119:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:    GluLeuThrGlnSerProGlyThrLeuSerLeuThrProGlyGluArg    151015    AlaIleLeuSerCysLysThrSerHisAsnIleTrpSerArgArgLeu    202530    AlaTrpTyrGlnLeuLysSerGlyGlnAlaProArgLeuLeuIleTyr    354045    GlyValSerLysArgAlaGlyGlyIleProAspArgPheSerGlySer    505560    GlySerAlaThrAspPheThrLeuThrIleSerArgValGluProGlu    65707580    AspPheAlaValTyrTyrCysGlnThrTyrGlyGlySerAlaTyrThr    859095    PheGlyGlnGlyThrLysLeuAspIleLysArg    100105    (2) INFORMATION FOR SEQ ID NO:120:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:    GluLeuThrGlnSerProGlyThrLeuSerLeuThrProGlyGluArg    151015    AlaIleLeuSerCysLysThrSerHisAsnIleTrpSerArgArgLeu    202530    AlaTrpTyrGlnLeuLysSerGlyGlnAlaProArgLeuLeuIleTyr    354045    GlyValSerLysArgAlaGlyGlyIleProAspArgPheSerGlySer    505560    GlySerAlaThrAspPheThrLeuThrIleSerArgValGluProGlu    65707580    AspPheAlaValTyrTyrCysGlnThrTyrGlyGlySerAlaTyrThr    859095    PheGlyGlnGlyThrLysLeuGluIleLysArg    100105    (2) INFORMATION FOR SEQ ID NO:121:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:    GluLeuThrGlnSerProGlyThrLeuSerSerThrProGlyGluArg    151015    AlaIleLeuSerCysLysThrSerHisAsnIleTrpSerArgArgLeu    202530    AlaTrpTyrGlnValLysSerGlyLeuProProArgLeuLeuIleHis    354045    GlyValSerArgArgAlaGlyGlyIleProAspArgPheSerGlySer    505560    GlySerAlaArgAspPheThrLeuThrIleSerArgLeuGluProAla    65707580    AspPheAlaValTyrTyrCysGlnThrTyrGlyGlySerSerTyrSer    859095    PheGlyGlnGlyThrLysLeuAspPheAsnArg    100105    (2) INFORMATION FOR SEQ ID NO:122:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:    GluLeuThrGlnSerProGlyThrLeuSerLeuAsnProGlyGluArg    151015    AlaValLeuSerCysArgThrSerArgAsnIleTrpSerArgArgLeu    202530    AlaTrpTyrGlnValArgArgGlyGlnAlaProArgLeuLeuIleHis    354045    GlyValSerLysArgAlaGlyGlyValProAspArgPheSerGlySer    505560    GlySerAlaArgAspPheThrLeuThrIleSerArgLeuGluProGlu    65707580    AspPheAlaValTyrPheCysGlnThrTyrGlyGlySerSerTyrThr    859095    PheGlyGlnGlyAsnLysLeuAspIleArgArg    100105    (2) INFORMATION FOR SEQ ID NO:123:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 126 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:    GlnValLysLeuLeuGluGlnSerGlyAlaGluValLysLysProGly    151015    AlaSerValLysValSerCysGlnAlaSerGlyTyrArgPheSerAsn    202530    PheValLeuHisTrpAlaArgGlnAlaProGlyHisArgProGluTrp    354045    MetGlyTrpIleAsnProAlaAsnGlyValThrGluIleProProLys    505560    PheGlnAspArgValSerLeuThrArgAspThrSerAlaGlyThrVal    65707580    TyrLeuGluLeuThrAsnLeuArgPheAlaAspThrAlaValTyrTyr    859095    CysAlaArgValGlyGluTrpThrTrpAspAspSerProGlnAspAsn    100105110    TyrTyrMetAspValTrpGlyLysGlyThrThrValThrVal    115120125    (2) INFORMATION FOR SEQ ID NO:124:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:    GlnValLysLeuLeuGluGlnSerGlyAlaGluValLysLysProGly    151015    AlaSerValLysValSerCysGlnAlaSerGlyTyrArgPheSerAsn    202530    PheValLeuHisTrpAlaArgGlnAlaProGlyHisArgProGluTrp    354045    MetGlyTrpIleAsnProAlaAsnGlyValThrGluIleSerProLys    505560    PheGlnAspArgValSerLeuThrGlyAspThrSerAlaSerThrVal    65707580    TyrLeuGluLeuArgAsnLeuArgPheAlaAspThrAlaValTyrTyr    859095    CysAlaArgValGlyGluTrpThrTrpAspAspSerProGlnAspAsn    100105110    TyrTyrMetAspValTrpGlyArgGlyThrThrValThr    115120125    (2) INFORMATION FOR SEQ ID NO:125:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 124 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:    GlnValLysLeuLeuGluGlnSerGlyAlaGluValLysLysProGly    151015    AlaSerValLysValSerCysGlnAlaSerGlyTyrArgPheSerAsn    202530    PheValLeuHisTrpAlaArgGlnAlaProGlyHisArgProGluTrp    354045    MetGlyTrpIleAsnProAlaAsnGlyValThrGluIleSerProLys    505560    PheGlnAspArgValSerLeuThrGlyAspThrSerAlaSerThrVal    65707580    TyrLeuGluLeuArgSerLeuArgPheAlaAspThrAlaValTyrTyr    859095    CysAlaArgValGlyGluTrpThrTrpAspAspSerProGlnAspAsn    100105110    TyrTyrMetAspValTrpGlyLysGlyThrThrVal    115120    (2) INFORMATION FOR SEQ ID NO:126:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 124 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:    GlnValLysLeuLeuGluGlnSerGlyAlaGluValLysLysProGly    151015    AlaSerValLysIleSerCysGlnAlaSerGlyTyrArgPheThrAsn    202530    PheValLeuHisTrpAlaArgGlnAlaProGlyGlnArgProGluTrp    354045    MetGlyTrpPheAsnProAlaAsnGlyIleLysGluIleSerProLys    505560    PheGlnAspArgValSerPheThrGlyAspThrSerAlaSerThrAla    65707580    TyrValGluLeuArgAsnLeuArgSerAlaAspThrAlaValTyrTyr    859095    CysAlaArgValGlyProTrpThrTrpAspAspSerProGlnAspAsn    100105110    TyrTyrMetAspValTrpGlyLysGlyThrThrVal    115120    (2) INFORMATION FOR SEQ ID NO:127:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 124 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:    GlnValLysLeuLeuGluGlnSerGlyAlaGluValLysLysProGly    151015    AlaSerValLysValSerCysGlnAlaSerGlyTyrArgPheSerAsn    202530    PheValLeuHisTrpAlaArgGlnAlaProGlyHisArgProGluTrp    354045    MetGlyTrpIleAsnProAlaAsnGlyValThrGluIleSerProLys    505560    PheGlnAspArgValSerLeuThrGlyAspThrSerAlaSerThrVal    65707580    TyrLeuGluLeuArgAsnLeuArgPheAlaAspThrAlaValTyrTyr    859095    CysAlaArgValGlyGluTrpThrTrpAspAspPheProGlnAspAsn    100105110    TyrTyrMetAspValTrpGlyLysGlyThrThrVal    115120    (2) INFORMATION FOR SEQ ID NO:128:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:    GlnValLysLeuLeuGluGlnSerGlyAlaGluValLysLysProGly    151015    AlaSerValLysLeuSerCysGlnAlaSerGlyTyrArgPheSerAsn    202530    PheValLeuHisTrpAlaArgGlnAlaProGlyHisArgProGluTrp    354045    MetGlyTrpIleAsnProAlaAsnGlyValThrGluIleSerProLys    505560    PheGlnAspArgValSerLeuThrGlyAspThrSerAlaSerThrVal    65707580    TyrLeuGluLeuArgAsnLeuArgPheAlaAspThrAlaValTyrTyr    859095    CysAlaArgValGlyGluTrpThrTrpAspAspSerProGlnAspAsn    100105110    TyrTyrMetAspValTrpGlyLysGlyThrThrValThr    115120125    (2) INFORMATION FOR SEQ ID NO:129:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:    GlnValLysLeuLeuGluGlnSerGlyThrGluValLysLysProGly    151015    AlaSerValLysIleSerCysLysAlaSerGlyTyrArgPheThrAsn    202530    PheProLeuHisTrpValArgGlnAlaProGlyGlnArgProGluTrp    354045    MetGlyTrpIleLysIleValAsnGlyGluLysLysTyrSerGlnLys    505560    PheValAspArgValThrPheThrGlyAspThrSerAlaAsnThrAla    65707580    TyrMetGluValArgGlyLeuArgSerAlaAspThrAlaThrTyrTyr    859095    CysAlaArgValGlyGluTrpThrTrpAspMetAspProGlnAlaAsn    100105110    TyrTyrMetAspValTrpGlyLysGlyThrThrValThr    115120125    (2) INFORMATION FOR SEQ ID NO:130:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 124 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:    GlnValLysLeuLeuGluGlnSerGlyAlaGluValLysLysProGly    151015    AlaSerValLysValSerCysGlnAlaSerGlyTyrArgPheSerAsn    202530    PheValIleHisTrpValArgGlnAlaProGlyGlnArgPheGluTrp    354045    MetGlyTrpIleAsnProTyrAsnGlyAsnLysGluPheSerAlaLys    505560    PheArgAspArgValThrPheThrAlaAspThrAspAlaAsnThrAla    65707580    TyrMetGluLeuArgSerLeuArgSerAlaAspThrAlaIleTyrTyr    859095    CysAlaArgValGlyProTyrThrTrpAspAspSerProGlnAspAsn    100105110    TyrTyrMetAspValTrpGlyLysGlyThrThrVal    115120    (2) INFORMATION FOR SEQ ID NO:131:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 124 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:    GlnValLysLeuLeuGluGlnSerGlyAlaGluValLysLysProGly    151015    AlaSerValLysValSerCysGlnAlaSerGlyTyrArgPheSerAsn    202530    PheValLeuHisTrpAlaArgGlnAlaProThrGlnAspLeuGluTrp    354045    MetGlyTrpIleAsnProAlaAsnGlyValLysGluIleSerProLys    505560    PheGlnAspArgValSerLeuThrGlyAspThrSerAlaSerThrVal    65707580    TyrLeuGluLeuArgSerLeuArgPheAlaAspThrAlaValTyrTyr    859095    CysAlaArgValGlyGluTrpThrTrpAspAspSerProGlnAspAsn    100105110    TyrTyrMetAspValTrpGlyLysGlyThrThrVal    115120    (2) INFORMATION FOR SEQ ID NO:132:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 124 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:    GlnValLysLeuLeuGluGlnSerGlyAlaGluValLysLysProGly    151015    AlaSerValLysValSerCysGlnAlaSerGlyTyrArgPheSerAsn    202530    PheValLeuHisTrpAlaArgGlnAlaProGlyHisArgProGluTrp    354045    MetGlyTrpIleAsnProAlaAsnGlyValThrGluIleProProLys    505560    PheGlnAspArgValSerLeuThrArgAspThrSerAlaGlyThrVal    65707580    TyrLeuGluLeuThrAsnLeuArgPheAlaAspThrAlaValTyrTyr    859095    CysAlaArgValGlyGluTrpThrTrpAspAspSerProGlnAspAsn    100105110    TyrTyrMetAspValTrpGlyLysGlyThrThrVal    115120    (2) INFORMATION FOR SEQ ID NO:133:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 37 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:    TCGAGGGTCGGTCGGTCTCTAGACGGTCGGTCGGTCA37    (2) INFORMATION FOR SEQ ID NO:134:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 37 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:    CTAGTGACCGACCGACCGTCTAGAGACCGACCGACCC37    (2) INFORMATION FOR SEQ ID NO:135:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:    CGGTCGGTCGGTCCTCGAGGGTCGGTCGGTCT32    (2) INFORMATION FOR SEQ ID NO:136:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:    CTAGAGACCGACCGACCCTCGAGGACCGACCGACCGAGCT40    (2) INFORMATION FOR SEQ ID NO:137:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:    CAAGGAGACAGGATCCATGAAATAC25    (2) INFORMATION FOR SEQ ID NO:138:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:    AGGGCGAATTGGATCCCGGGCCCCC25    (2) INFORMATION FOR SEQ ID NO:139:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:    CTAGTCATCATCATCATCATTAAGCTAGC29    (2) INFORMATION FOR SEQ ID NO:140:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:    CTAGGCTAGCTTAATGATGATGATGATGA29    (2) INFORMATION FOR SEQ ID NO:141:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /label=J    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13    (D) OTHER INFORMATION: /label=ZC    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:    SerIleSerIleGlyProGlyArgAlaPheTyrThrGly    1510    (2) INFORMATION FOR SEQ ID NO:142:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 126 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:    LeuLeuGluSerGlyProGlyLeuValLysProSerGluThrLeuSer    151015    LeuThrCysThrValSerGlyGlySerLeuSerSerPheAspTrpAsn    202530    TrpIleArgGlnProAlaGlyLysGlyLeuGluTrpIleGlyArgIle    354045    TyrProSerGlyAsnThrHisTyrAsnProSerLeuArgSerArgVal    505560    ThrMetSerArgAspThrSerLysAsnGlnPheSerValLysLeuThr    65707580    SerValThrAlaAlaAspThrAlaLeuTyrTyrCysAlaArgGluAsn    859095    ThrGlyArgThrIleGluGluIleGlyAsnPhePheAspIleTrpGly    100105110    GlnGlyThrLeuValThrValSerSerAlaSerThrLysGly    115120125    (2) INFORMATION FOR SEQ ID NO:143:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 122 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:    LeuLeuLysSerGlyGlyGlyLeuValLysProGlyGlySerLeuArg    151015    LeuSerCysValIleSerAlaPheSerPheSerGlyTyrAsnIleAsn    202530    TrpValArgGlnAlaProGlyLysGlyLeuGluTrpValSerSerIle    354045    SerMetSerThrGlySerLeuSerTyrAlaAspSerMetLysGlyArg    505560    PheThrIleSerArgAspAsnAlaLysAsnSerValTyrLeuGluMet    65707580    SerSerLeuThrAlaGluAspThrAlaMetTyrTyrCysAlaAlaArg    859095    ThrProLeuValGlyArgAlaLeuAspIleTrpGlyGlnGlyThrVal    100105110    ValThrValSerSerAlaSerThrLysGly    115120    (2) INFORMATION FOR SEQ ID NO:144:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 132 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:    LeuLeuGluSerGlyGlyGlyLeuValLysProGlyGlySerLeuArg    151015    LeuSerCysSerAlaSerGlyPheThrPheSerSerTyrGlyMetAsn    202530    TrpValArgGlnAlaProGlyLysGlyProGluTrpValAlaTyrIle    354045    SerSerSerArgLysTyrThrGluTyrAlaAspSerValLysGlyArg    505560    PheThrIleSerArgGluAsnAlaLysTyrSerValPheLeuGlnLeu    65707580    AspSerLeuThrAlaGluAspThrAlaIleTyrTyrCysAlaArgGly    859095    ArgAspPheTyrSerGlyPheGlyArgArgAspAspPheHisLeuHis    100105110    TyrMetAspValTrpGlyLysGlyThrThrValThrValSerSerAla    115120125    SerThrLysGly    130    (2) INFORMATION FOR SEQ ID NO:145:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 126 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:    LeuLeuGluGlnSerGlyGlyGlyLeuValGlnProGlyGlySerLeu    151015    ArgIleSerCysValAlaSerGlyAspIlePheTyrSerTyrAlaMet    202530    SerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpValAlaSer    354045    IleSerGlyThrGlyGlySerAsnTyrTyrAlaAspSerValLysGly    505560    ArgPheThrIleSerArgAspAsnSerLysSerThrLeuTyrLeuGln    65707580    MetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCysAlaArg    859095    AspArgGlyProArgIleGlyIleArgGlyTrpPheAspSerTrpGly    100105110    GlnGlyThrLeuValThrValSerSerAlaSerThrLysGly    115120125    (2) INFORMATION FOR SEQ ID NO:146:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 124 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:    LeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGlySerLeuArg    151015    LeuSerCysAlaAlaSerGlyPheLeuTyrSerSerPheAlaMetSer    202530    TrpValArgGlnAlaProGlyLysGlyLeuAlaTrpValSerThrIle    354045    SerAlaSerGlyGlySerThrLysTyrAlaAspSerValLysGlyArg    505560    PheIleIleSerArgAspAsnSerLysAsnThrIleTyrLeuGlnMet    65707580    AspSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaLysAsn    859095    PheArgAlaPheAlaArgAspProTrpGlyAspTrpGlyGlnGlyThr    100105110    LeuValThrValSerSerAlaSerAlaSerThrLys    115120    (2) INFORMATION FOR SEQ ID NO:147:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 109 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:    MetAlaGluLeuThrGlnSerProGlyThrLeuSerLeuSerProGly    151015    GluArgValIleValSerCysArgAlaSerGlnSerValSerSerAsn    202530    TyrLeuAlaTrpTyrGlnGlnLysProGlyGlnAlaProArgLeuLeu    354045    IleTyrGlyAlaSerAsnArgAlaThrGlyIleProAspArgPheSer    505560    GlySerGlySerGlyThrAspPheThrLeuThrIleSerArgLeuGlu    65707580    ProGluAspPheAlaValTyrTyrCysGlnGlnTyrGlySerSerGly    859095    ThrPheGlyGlnGlyThrLysValGluIleLysArgThr    100105    (2) INFORMATION FOR SEQ ID NO:148:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 112 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:    MetAlaGluLeuThrGlnSerProGlyThrLeuSerLeuSerProGly    151015    GluArgAlaThrPheSerCysArgSerSerHisSerIleHisThrArg    202530    ArgValAlaTrpTyrGlnHisLysProGlyGlnAlaProArgLeuVal    354045    IleHisGlyValSerAsnArgAlaSerGlyIleSerAspArgPheSer    505560    GlySerGlySerGlyThrAspPheThrLeuThrIleThrArgValGlu    65707580    ProGluAspPheAlaLeuTyrTyrCysGlnValTyrGlyAlaSerSer    859095    TyrThrPheGlyGlnGlyThrLysLeuGluArgLysArgThrValVal    100105110    (2) INFORMATION FOR SEQ ID NO:149:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 111 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:    MetAlaGluLeuThrGlnSerProGlyThrLeuSerLeuSerProGly    151015    GluArgAlaThrLeuSerCysArgAlaSerGlnSerValSerAsnGly    202530    TyrLeuAlaTrpTyrGlnGlnLysProGlyGlnAlaProArgLeuLeu    354045    IleTyrGlyAlaSerThrArgAlaThrAspIleProAspArgPheSer    505560    GlySerGlySerGlyAlaAspPheThrLeuAlaIleSerArgLeuGlu    65707580    ProGluAspPheAlaValTyrTyrCysGlnGlnTyrAlaGlySerHis    859095    ThrPheGlyGlnGlyThrLysLeuGluIleLysArgThrValAla    100105110    (2) INFORMATION FOR SEQ ID NO:150:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 111 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:    MetAlaGluLeuThrGlnSerProSerSerLeuSerAlaSerValGly    151015    AspArgValThrIleThrCysArgProSerGlnGlyIleGlyArgPhe    202530    PheAsnTrpTyrGlnGlnLysProGlyLysAlaProAsnLeuLeuIle    354045    TyrAlaAlaAspIleLeuGlnSerGlyValProSerArgPheSerGly    505560    SerGlySerGlyThrAspPheThrLeuThrIleSerSerLeuGlnPro    65707580    GluAspPheAlaThrTyrTyrCysGlnGlnSerTyrSerThrProTyr    859095    ThrPheGlyGlnGlyThrArgLeuAspIleLysArgThrValAla    100105110    (2) INFORMATION FOR SEQ ID NO:151:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 112 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:    MetAlaGluLeuThrGlnSerProSerSerLeuSerAlaSerValGly    151015    AspArgValThrIleThrCysArgAlaSerGlnGlyValSerSerSer    202530    TyrLeuAlaTrpTyrGlnGlnLysProGlyGlnAlaProArgLeuVal    354045    IlePheGlyAlaTyrSerArgAlaThrGlyIleProAspArgPheSer    505560    GlySerGlySerGlyThrAspPheThrLeuThrIleSerArgLeuGlu    65707580    ProGluAspPheAlaValTyrTyrCysGlnGlnTyrGlySerSerPro    859095    IleThrPheGlyProGlyThrLysValAspIleLysArgThrValAla    100105110    (2) INFORMATION FOR SEQ ID NO:152:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 729 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 9..715    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:    AGCTTACCATGGGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGG50    MetGlyValProThrGlnValLeuGlyLeuLeuLeuLeuTrp    1510    CTTACAGATGCCAGATGTGAGATCGTTCTCACGCAGTCTCCAGGCACC98    LeuThrAspAlaArgCysGluIleValLeuThrGlnSerProGlyThr    15202530    CTGTCTCTGTCTCCAGGGGAAAGAGCCACCTTCTCCTGTAGGTCCAGT146    LeuSerLeuSerProGlyGluArgAlaThrPheSerCysArgSerSer    354045    CACAGCATTCGCAGCCGCCGCGTAGCCTGGTACCAGCACAAACCTGGC194    HisSerIleArgSerArgArgValAlaTrpTyrGlnHisLysProGly    505560    CAGGCTCCAAGGCTGGTCATACATGGTGTTTCCAATAGGGCCTCTGGC242    GlnAlaProArgLeuValIleHisGlyValSerAsnArgAlaSerGly    657075    ATCTCAGACAGGTTCAGCGGCAGTGGGTCTGGGACAGACTTCACTCTC290    IleSerAspArgPheSerGlySerGlySerGlyThrAspPheThrLeu    808590    ACCATCACCAGAGTGGAGCCTGAAGACTTTGCACTGTACTACTGTCAG338    ThrIleThrArgValGluProGluAspPheAlaLeuTyrTyrCysGln    95100105110    GTCTATGGTGCCTCCTCGTACACTTTTGGCCAGGGGACCAAACTGGAG386    ValTyrGlyAlaSerSerTyrThrPheGlyGlnGlyThrLysLeuGlu    115120125    AGGAAACGAACTGTGCCTGCACCATCTGTCTTCATCTTCCCGCCATCT434    ArgLysArgThrValProAlaProSerValPheIlePheProProSer    130135140    GATGAGCAGTTGAAATCTGGGACTGCCTCTGTTGTGTGCCTGCTGAAT482    AspGluGlnLeuLysSerGlyThrAlaSerValValCysLeuLeuAsn    145150155    AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC530    AsnPheTyrProArgGluAlaLysValGlnTrpLysValAspAsnAla    160165170    CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG578    LeuGlnSerGlyAsnSerGlnGluSerValThrGluGlnAspSerLys    175180185190    GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGAC626    AspSerThrTyrSerLeuSerSerThrLeuThrLeuSerLysAlaAsp    195200205    TACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG674    TyrGluLysHisLysValTyrAlaCysGluValThrHisGlnGlyLeu    210215220    AGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAATTCTAGAGA725    SerSerProValThrLysSerPheAsnArgGlyGluCys    225230235    ATTC729    (2) INFORMATION FOR SEQ ID NO:153:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 235 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:    MetGlyValProThrGlnValLeuGlyLeuLeuLeuLeuTrpLeuThr    151015    AspAlaArgCysGluIleValLeuThrGlnSerProGlyThrLeuSer    202530    LeuSerProGlyGluArgAlaThrPheSerCysArgSerSerHisSer    354045    IleArgSerArgArgValAlaTrpTyrGlnHisLysProGlyGlnAla    505560    ProArgLeuValIleHisGlyValSerAsnArgAlaSerGlyIleSer    65707580    AspArgPheSerGlySerGlySerGlyThrAspPheThrLeuThrIle    859095    ThrArgValGluProGluAspPheAlaLeuTyrTyrCysGlnValTyr    100105110    GlyAlaSerSerTyrThrPheGlyGlnGlyThrLysLeuGluArgLys    115120125    ArgThrValProAlaProSerValPheIlePheProProSerAspGlu    130135140    GlnLeuLysSerGlyThrAlaSerValValCysLeuLeuAsnAsnPhe    145150155160    TyrProArgGluAlaLysValGlnTrpLysValAspAsnAlaLeuGln    165170175    SerGlyAsnSerGlnGluSerValThrGluGlnAspSerLysAspSer    180185190    ThrTyrSerLeuSerSerThrLeuThrLeuSerLysAlaAspTyrGlu    195200205    LysHisLysValTyrAlaCysGluValThrHisGlnGlyLeuSerSer    210215220    ProValThrLysSerPheAsnArgGlyGluCys    225230235    (2) INFORMATION FOR SEQ ID NO:154:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3282 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 15..452    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154:    AATTCGCCGCCACCATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCA50    MetGluTrpSerTrpValPheLeuPhePheLeuSer    1510    GTAACTACAGGTGTCCACTCCCAGGTTCAGCTGGTTCAGTCCGGGGCT98    ValThrThrGlyValHisSerGlnValGlnLeuValGlnSerGlyAla    152025    GAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCTTGTCAGGCTTCT146    GluValLysLysProGlyAlaSerValLysValSerCysGlnAlaSer    303540    GGATACAGATTCAGTAACTTTGTTATTCATTGGGTGCGCCAGGCCCCC194    GlyTyrArgPheSerAsnPheValIleHisTrpValArgGlnAlaPro    45505560    GGACAGAGGTTTGAGTGGATGGGATGGATCAATCCTTACAACGGAAAC242    GlyGlnArgPheGluTrpMetGlyTrpIleAsnProTyrAsnGlyAsn    657075    AAAGAATTTTCAGCGAAGTTCCAGGACAGAGTCACCTTTACCGCGGAC290    LysGluPheSerAlaLysPheGlnAspArgValThrPheThrAlaAsp    808590    ACATCCGCGAACACAGCCTACATGGAGTTGAGGAGCCTCAGGTCTGCA338    ThrSerAlaAsnThrAlaTyrMetGluLeuArgSerLeuArgSerAla    95100105    GACACGGCTGTTTATTATTGTGCGAGAGTGGGGCCATATAGTTGGGAT386    AspThrAlaValTyrTyrCysAlaArgValGlyProTyrSerTrpAsp    110115120    GATTCTCCCCAGGACAATTATTATATGGACGTCTGGGGCAAAGGAACC434    AspSerProGlnAspAsnTyrTyrMetAspValTrpGlyLysGlyThr    125130135140    ACGGTCATCGTGAGCTCAGCTTCCACCAAGGGCCCATCGGTCTTCCCC482    ThrValIleValSerSer    145    CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG542    GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTG602    CACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC662    GTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGC722    AACACCAAGGTGGACAAGAAAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCT782    GGAAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGCCCCAGTCCAGGG842    CAGCAAGGCAGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCT902    CAGGGAGAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCC962    TAACCCAGGCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATAT1022    CCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCA1082    GCTCGGACACCTTCTCTCCTCCCAGATTCGAGTAACTCCCAATCTTCTCTCTGCAGAGCC1142    CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGCCCAGGCCTC1202    GCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCC1262    AGCCGGGTGCTGACACGTCCACCTCCATCTCTCCCTCAGCACCTGAGGCCGCGGGAGGAC1322    CATCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG1382    AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT1442    ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACA1502    GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGG1562    AGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCA1622    AAGCCAAAGGTGGGACCCGTGGGGTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCA1682    CCCTCTGCCCTGAGAGTGACCGCTGTACCAACCTCTGTCCCTACAGGGCAGCCCCGAGAA1742    CCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG1802    ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG1862    CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC1922    CTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC1982    TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG2042    GGTAAATGAGTGCGACGGCCGGCAAGCCCCCGCTCCCCGGGCTCTCGCGGTCGCACGAGG2102    ATGCTTGGCACGTACCCCCTGTACATACTTCCCGGGCGCCCAGCATGGAAATAAAGCACC2162    CAGCGCTGCCCTGGGCCCCTGCGAGACTGTGATGGTTCTTTCCACGGGTCAGGCCGAGTC2222    TGAGGCCTGAGTGGCATGAGGGAGGCAGAGCGGGTCCCACTGTCCCCACACTGGCCCAGG2282    CTGTGCAGGTGTGCCTGGGCCGCCTAGGGTGGGGCTCAGCCAGGGGCTGCCCTCGGCAGG2342    GTGGGGGATTTGCCAGCGTTGCCCTCCCTCCAGCAGCACCTGCCCTGGGCTGGGCCACGG2402    GAAGCCCTAGGAGCCCCTGGGGACAGACACACAGCCCCTGCCTCTGTAGGAGACTGTCCT2462    GTTCTGTGAGCGCCCTGTCCTCCGACCTCCATGCCCACTCGGGGGCATGCCTAGTCCATG2522    TGCGTAGGGACAGGCCCTCCCTCACCCATCTACCCCCACGGCACTAACCCCTGGCTGTCC2582    TGCCCAGCCTCGCACCCGCATGGGGACACAACCGACTCCGGGGACATGCACTCTCGGGCC2642    CTGTGGAGGGACTGGTGCAGATGCCCACACACACACTCAGTCCAGACCCGTTCAACAAAA2702    CCCCCGCACTGAGGTTGGCCGGCCACACGGCCACCACACACACACGTGCACGCCTCACAC2762    ACGGAGCCTCACCCGGGCGAACTGCACAGCACCCAGACCAGAGCAAGGTCCTCGCACACG2822    TGAACACTCCTCGGACACAGGCCCCCACGAGCCCCACGCGGCACCTCAAGGCCCACGAGC2882    CTCTCGGCAGCTTCTCCACATGCTGACCTGCTCAGACAAACCCAGCCCTCCTCTCACAAG2942    GGTGCCCCTGCAGCCGCCACACACACACAGGGGATCACACACCACGTCACGTCCCTGGCC3002    CTGGCCCACTTCCCAGTGCCGCCCTTCCCTGCAGGGCGGATCATAATCAGCCATACCACA3062    TTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACAT3122    AAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAA3182    AGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGT3242    TTGTCCAAACTCATCAATGTATCTTATCATGTCTAGATCC3282    (2) INFORMATION FOR SEQ ID NO:155:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 146 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:    MetGluTrpSerTrpValPheLeuPhePheLeuSerValThrThrGly    151015    ValHisSerGlnValGlnLeuValGlnSerGlyAlaGluValLysLys    202530    ProGlyAlaSerValLysValSerCysGlnAlaSerGlyTyrArgPhe    354045    SerAsnPheValIleHisTrpValArgGlnAlaProGlyGlnArgPhe    505560    GluTrpMetGlyTrpIleAsnProTyrAsnGlyAsnLysGluPheSer    65707580    AlaLysPheGlnAspArgValThrPheThrAlaAspThrSerAlaAsn    859095    ThrAlaTyrMetGluLeuArgSerLeuArgSerAlaAspThrAlaVal    100105110    TyrTyrCysAlaArgValGlyProTyrSerTrpAspAspSerProGln    115120125    AspAsnTyrTyrMetAspValTrpGlyLysGlyThrThrValIleVal    130135140    SerSer    145    (2) INFORMATION FOR SEQ ID NO:156:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13254 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: circular    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:    TTCATTGATCATTAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACC60    TCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGT120    TTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAG180    CATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATG240    TCTGGATCTCTAGCTTCGTGTCAAGGACGGTGACTGCAGTGAATAATAAAATGTGTGTTT300    GTCCGAAATACGCGTTTTGAGATTTCTGTCGCCGACTAAATTCATGTCGCGCGATAGTGG360    TGTTTATCGCCGATAGAGATGGCGATATTGGAAAAATCGATATTTGAAAATATGGCATAT420    TGAAAATGTCGCCGATGTGAGTTTCTGTGTAACTGATATCGCCATTTTTCCAAAAGTGAT480    TTTTGGGCATACGCGATATCTGGCGATAGCGCTTATATCGTTTACGGGGGATGGCGATAG540    ACGACTTTGGTGACTTGGGCGATTCTGTGTGTCGCAAATATCGCAGTTTCGATATAGGTG600    ACAGACGATATGAGGCTATATCGCCGATAGAGGCGACATCAAGCTGGCACATGGCCAATG660    CATATCGATCTATACATTGAATCAATATTGGCCATTAGCCATATTATTCATTGGTTATAT720    AGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCCATATCATAATATGTA780    CATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTA840    TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTAC900    ATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTC960    AATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGT1020    GGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTAC1080    GCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGAC1140    CTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGT1200    GATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCC1260    AAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTT1320    TCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTG1380    GGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCC1440    ACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACG1500    GTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTA1560    TAGGCCCACCCCCTTGGCTTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTCTATACA1620    CCCCCGCTTCCTCATGTTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGA1680    CCATTATTGACCACTCCCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGC1740    TCTTTGCCACAACTCTCTTTATTGGCTATATGCCAATACACTGTCCTTCAGAGACTGACA1800    CGGACTCTGTATTTTTACAGGATGGGGTCTCATTTATTATTTACAAATTCACATATACAA1860    CACCACCGTCCCCAGTGCCCGCAGTTTTTATTAAACATAACGTGGGATCTCCACGCGAAT1920    CTCGGGTACGTGTTCCGGACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCTACATCCGA1980    GCCCTGCTCCCATGCCTCCAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGT2040    GGAGGCCAGACTTAGGCACAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGGCCGT2100    GGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGGGAGCGGGCTTGCACCGCTGACGCATT2160    TGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATA2220    AGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGC2280    AGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTT2340    CCTTTCCATGGGTCTTTTCTGCAGTCACCGTCCTTGACACGAAGCTTGGGCTGCAGGTCG2400    ATCGACTCTAGAGGATCGATCCCCGGGCGAGCTCGAATTCGCCGCCACCATGGAATGGAG2460    CTGGGTCTTTCTCTTCTTCCTGTCAGTAACTACAGGTGTCCACTCCCAGGTTCAGCTGGT2520    TCAGTCCGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCTTGTCAGGCTTC2580    TGGATACAGATTCAGTAACTTTGTTATTCATTGGGTGCGCCAGGCCCCCGGACAGAGGTT2640    TGAGTGGATGGGATGGATCAATCCTTACAACGGAAACAAAGAATTTTCAGCGAAGTTCCA2700    GGACAGAGTCACCTTTACCGCGGACACATCCGCGAACACAGCCTACATGGAGTTGAGGAG2760    CCTCAGGTCTGCAGACACGGCTGTTTATTATTGTGCGAGAGTGGGGCCATATAGTTGGGA2820    TGATTCTCCCCAGGACAATTATTATATGGACGTCTGGGGCAAAGGAACCACGGTCATCGT2880    GAGCTCAGCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC2940    CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGAC3000    GGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA3060    GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC3120    CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGT3180    TGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTG3240    CCTGGACGCATCCCGGCTATGCAGCCCCAGTCCAGGGCAGCAAGGCAGGCCCCGTCTGCC3300    TCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGGGTCTTCTGGCTTT3360    TTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAGGCCCTGCACACAAAG3420    GGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGGGAGGACCCTGCCCCTGACC3480    TAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACCTTCTCTCCTCCC3540    AGATTCGAGTAACTCCCAATCTTCTCTCTGCAGAGCCCAAATCTTGTGACAAAACTCACA3600    CATGCCCACCGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACA3660    GGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACACGTCCACC3720    TCCATCTCTCCCTCAGCACCTGAGGCCGCGGGAGGACCATCAGTCTTCCTCTTCCCCCCA3780    AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGAC3840    GTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT3900    AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC3960    CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC4020    AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGG4080    GTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGC4140    TGTACCAACCTCTGTCCCTACAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC4200    ATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA4260    TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC4320    CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGA4380    CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA4440    CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGGC4500    AAGCCCCCGCTCCCCGGGCTCTCGCGGTCGCACGAGGATGCTTGGCACGTACCCCCTGTA4560    CATACTTCCCGGGCGCCCAGCATGGAAATAAAGCACCCAGCGCTGCCCTGGGCCCCTGCG4620    AGACTGTGATGGTTCTTTCCACGGGTCAGGCCGAGTCTGAGGCCTGAGTGGCATGAGGGA4680    GGCAGAGCGGGTCCCACTGTCCCCACACTGGCCCAGGCTGTGCAGGTGTGCCTGGGCCGC4740    CTAGGGTGGGGCTCAGCCAGGGGCTGCCCTCGGCAGGGTGGGGGATTTGCCAGCGTTGCC4800    CTCCCTCCAGCAGCACCTGCCCTGGGCTGGGCCACGGGAAGCCCTAGGAGCCCCTGGGGA4860    CAGACACACAGCCCCTGCCTCTGTAGGAGACTGTCCTGTTCTGTGAGCGCCCTGTCCTCC4920    GACCTCCATGCCCACTCGGGGGCATGCCTAGTCCATGTGCGTAGGGACAGGCCCTCCCTC4980    ACCCATCTACCCCCACGGCACTAACCCCTGGCTGTCCTGCCCAGCCTCGCACCCGCATGG5040    GGACACAACCGACTCCGGGGACATGCACTCTCGGGCCCTGTGGAGGGACTGGTGCAGATG5100    CCCACACACACACTCAGTCCAGACCCGTTCAACAAAACCCCCGCACTGAGGTTGGCCGGC5160    CACACGGCCACCACACACACACGTGCACGCCTCACACACGGAGCCTCACCCGGGCGAACT5220    GCACAGCACCCAGACCAGAGCAAGGTCCTCGCACACGTGAACACTCCTCGGACACAGGCC5280    CCCACGAGCCCCACGCGGCACCTCAAGGCCCACGAGCCTCTCGGCAGCTTCTCCACATGC5340    TGACCTGCTCAGACAAACCCAGCCCTCCTCTCACAAGGGTGCCCCTGCAGCCGCCACACA5400    CACACAGGGGATCACACACCACGTCACGTCCCTGGCCCTGGCCCACTTCCCAGTGCCGCC5460    CTTCCCTGCAGGGCGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTT5520    AAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGT5580    TAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCAC5640    AAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATC5700    TTATCATGTCTAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGG5760    TGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTT5820    CGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCGTGGCCGGGGGACTG5880    TTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAAC5940    CTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACCTCGGGCC6000    GCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC6060    TCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA6120    AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT6180    CTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTG6240    TAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGC6300    GCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTG6360    GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTC6420    TTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTG6480    CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC6540    GCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT6600    CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGT6660    TAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAA6720    AAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAA6780    TGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCC6840    TGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCT6900    GCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCA6960    GCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATT7020    AATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT7080    GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCC7140    GGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGC7200    TCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTT7260    ATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACT7320    GGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGC7380    CCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATT7440    GGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCG7500    ATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCT7560    GGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAA7620    TGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGT7680    CTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGC7740    ACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACC7800    TATAAAAATAGGCGTATCACGAGGCCCTGATGGCTCTTTGCGGCACCCATCGTTCGTAAT7860    GTTCCGTGGCACCGAGGACAACCCTCAAGAGAAAATGTAATCACACTGGCTCACCTTCGG7920    GTGGGCCTTTCTGCGTTTATAAGGAGACACTTTATGTTTAAGAAGGTTGGTAAATTCCTT7980    GCGGCTTTGGCAGCCAAGCTAGATCCGGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAA8040    GTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAAC8100    CAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAG8160    TCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGC8220    CCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGC8280    TATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAACTAGCTTG8340    GGGCCACCGCTCAGAGCACCTTCCACCATGGCCACCTCAGCAAGTTCCCACTTGAACAAA8400    AACATCAAGCAAATGTACTTGTGCCTGCCCCAGGGTGAGAAAGTCCAAGCCATGTATATC8460    TGGGTTGATGGTACTGGAGAAGGACTGCGCTGCAAAACCCGCACCCTGGACTGTGAGCCC8520    AAGTGTGTAGAAGAGTTACCTGAGTGGAATTTTGATGGCTCTAGTACCTTTCAGTCTGAG8580    GGCTCCAACAGTGACATGTATCTCAGCCCTGTTGCCATGTTTCGGGACCCCTTCCGCAGA8640    GATCCCAACAAGCTGGTGTTCTGTGAAGTTTTCAAGTACAACCGGAAGCCTGCAGAGACC8700    AATTTAAGGCACTCGTGTAAACGGATAATGGACATGGTGAGCAACCAGCACCCCTGGTTT8760    GGAATGGAACAGGAGTATACTCTGATGGGAACAGATGGGCACCCTTTTGGTTGGCCTTCC8820    AATGGCTTTCCTGGGCCCCAAGGTCCGTATTACTGTGGTGTGGGCGCAGACAAAGCCTAT8880    GGCAGGGATATCGTGGAGGCTCACTACCGCGCCTGCTTGTATGCTGGGGTCAAGATTACA8940    GGAACAAATGCTGAGGTCATGCCTGCCCAGTGGGAACTCCAAATAGGACCCTGTGAAGGA9000    ATCCGCATGGGAGATCATCTCTGGGTGGCCCGTTTCATCTTCATCGAGTATGTGAAGACT9060    TTGGGGTAATAGCAACCTTTGACCCCAAGCCCATTCCTGGGAACTGGAATGGTGCAGGCT9120    GCCATACCAACTTTAGCACCAAGGCCATGCGGGAGGAGAATGGTCTGAAGCACATCGAGG9180    AGGCCATCGAGAAACTAAGCAAGCGGCACCGGTACCACATTCGAGCCTACGATCCCAAGG9240    GGGGCCTGGACAATGCCCGTGGTCTGACTGGGTTCCACGAAACGTCCAACATCAACGACT9300    TTTCTGCTGGTGTCGCCAATCGCAGTGCCAGCATCCGCATTCCCCGGACTGTCGGCCAGG9360    AGAAGAAAGGTTACTTTGAAGACCGCGGCCCCTCTGCCAATTGTGACCCCTTTGCAGTGA9420    CAGAAGCCATCGTCCGCACATGCCTTCTCAATGAGACTGGCCACGAGCCCTTCCAATACA9480    AAAACTAATTAGACTTTGAGTGATCTTGAGCCTTTCCTAGTTCATCCCACCCCGCCCCAG9540    AGAGATCTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCTAC9600    AGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACT9660    GATTCTAATTGTTTGTGTATTTTAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTG9720    GTGGAATGCCTTTAATGAGGAAAACCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGA9780    TGAGGCTACTGCTGACTCTCAACATTCTACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGA9840    CCCCAAGGACTTTCCTTCAGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAG9900    AACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCACTGCTATACAAGAA9960    AATTATGGAAAAATATTCTGTAACCTTTATAAGTAGGCATAACAGTTATAATCATAACAT10020    ACTGTTTTTTCTTACTCCACACAGGCATAGAGTGTCTGCTATTAATAACTATGCTCAAAA10080    ATTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAATATTTGATGTATAG10140    TGCCTTGACTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAA10200    AAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTA10260    ACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAA10320    ATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTT10380    ATCATGTCTGGATCTCTAGCTTCGTGTCAAGGACGGTGACTGCAGTGAATAATAAAATGT10440    GTGTTTGTCCGAAATACGCGTTTTGAGATTTCTGTCGCCGACTAAATTCATGTCGCGCGA10500    TAGTGGTGTTTATCGCCGATAGAGATGGCGATATTGGAAAAATCGATATTTGAAAATATG10560    GCATATTGAAAATGTCGCCGATGTGAGTTTCTGTGTAACTGATATCGCCATTTTTCCAAA10620    AGTGATTTTTGGGCATACGCGATATCTGGCGATAGCGCTTATATCGTTTACGGGGGATGG10680    CGATAGACGACTTTGGTGACTTGGGCGATTCTGTGTGTCGCAAATATCGCAGTTTCGATA10740    TAGGTGACAGACGATATGAGGCTATATCGCCGATAGAGGCGACATCAAGCTGGCACATGG10800    CCAATGCATATCGATCTATACATTGAATCAATATTGGCCATTAGCCATATTATTCATTGG10860    TTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCCATATCATAA10920    TATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGAC10980    TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCG11040    CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT11100    GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCA11160    ATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC11220    AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA11280    CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTAC11340    CATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGG11400    ATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACG11460    GGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGT11520    ACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACG11580    CCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCG11640    GGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAG11700    AGTCTATAGGCCCACCCCCTTGGCTTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTC11760    TATACACCCCCGCTTCCTCATGTTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGT11820    TATTGACCATTATTGACCACTCCCCTATTGGTGACGATACTTTCCATTACTAATCCATAA11880    CATGGCTCTTTGCCACAACTCTCTTTATTGGCTATATGCCAATACACTGTCCTTCAGAGA11940    CTGACACGGACTCTGTATTTTTACAGGATGGGGTCTCATTTATTATTTACAAATTCACAT12000    ATACAACACCACCGTCCCCAGTGCCCGCAGTTTTTATTAAACATAACGTGGGATCTCCAC12060    GCGAATCTCGGGTACGTGTTCCGGACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCTAC12120    ATCCGAGCCCTGCTCCCATGCCTCCAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCT12180    AACAGTGGAGGCCAGACTTAGGCACAGCACGATGCCCACCACCACCAGTGTGCCGCACAA12240    GGCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGGGAGCGGGCTTGCACCGCTGA12300    CGCATTTGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTT12360    CTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGT12420    CTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAG12480    ACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCGTCCTTGACACGAAGCTTACCATGG12540    GTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGATGCCAGATGTGAGA12600    TCGTTCTCACGCAGTCTCCAGGCACCCTGTCTCTGTCTCCAGGGGAAAGAGCCACCTTCT12660    CCTGTAGGTCCAGTCACAGCATTCGCAGCCGCCGCGTAGCCTGGTACCAGCACAAACCTG12720    GCCAGGCTCCAAGGCTGGTCATACATGGTGTTTCCAATAGGGCCTCTGGCATCTCAGACA12780    GGTTCAGCGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCACCAGAGTGGAGCCTG12840    AAGACTTTGCACTGTACTACTGTCAGGTCTATGGTGCCTCCTCGTACACTTTTGGCCAGG12900    GGACCAAACTGGAGAGGAAACGAACTGTGCCTGCACCATCTGTCTTCATCTTCCCGCCAT12960    CTGATGAGCAGTTGAAATCTGGGACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATC13020    CCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG13080    AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC13140    TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCC13200    TGAGATCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAATTCTAGAGAA13254    (2) INFORMATION FOR SEQ ID NO:157:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:    CAGGTTCAGCTGGTTCAGTCCGGGGCT27    (2) INFORMATION FOR SEQ ID NO:158:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 44 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:158:    CCTTGGAGCTCACGATGACCGTGGTTCCTTGGCCCCAGACGTCC44    (2) INFORMATION FOR SEQ ID NO:159:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 60 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:    GGCCGCGAATTCGCCGCCACCATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTA60    (2) INFORMATION FOR SEQ ID NO:160:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:    AGCCCCGGACTGAACCAGCTGAACCTG27    (2) INFORMATION FOR SEQ ID NO:161:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:    GGAGTTGAGGAGCCTCAGGTCTGCAGACACGG32    (2) INFORMATION FOR SEQ ID NO:162:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:    CCGTGTCTGCAGACCTGTGGCTCCTCAACTCC32    (2) INFORMATION FOR SEQ ID NO:163:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:    GATGCCAGATGTGAGATCGTTCTCACGCAGTCT33    (2) INFORMATION FOR SEQ ID NO:164:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 67 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164:    GCGGGATCCGAATTCTCTAGAATTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCG60    AACTCAG67    (2) INFORMATION FOR SEQ ID NO:165:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 51 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:    GCGCGAATTCACCATGGGTGTGCCCACTCAGGTCCTGGGGGTTGCTGCTGC51    (2) INFORMATION FOR SEQ ID NO:166:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:    AGACTGCGTGAGAACGATCTCACATCTGGCATC33    (2) INFORMATION FOR SEQ ID NO:167:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 50 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:167:    GCGCAAGCTTACCATGGGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGC50    (2) INFORMATION FOR SEQ ID NO:168:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 729 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168:    GAATTCTCTAGAATTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCGAACTCAGGC60    CCTGATGGGTGACTTCGCAGGCGTAGACTTTGTGTTTCTCGTAGTCTGCTTTGCTCAGCG120    TCAGGGTGCTGCTGAGGCTGTAGGTGCTGTCCTTGCTGTCCTGCTCTGTGACACTCTCCT180    GGGAGTTACCCGATTGGAGGGCGTTATCCACCTTCCACTGTACTTTGGCCTCTCTGGGAT240    AGAAGTTATTCAGCAGGCACACAACAGAGGCAGTCCCAGATTTCAACTGCTCATCAGATG300    GCGGGAAGATGAAGACAGATGGTGCAGGCACAGTTCGTTTCCTCTCCAGTTTGGTCCCCT360    GGCCAAAAGTGTACGAGGAGGCACCATAGACCTGACAGTAGTACAGTGCAAAGTCTTCAG420    GCTCCACTCTGGTGATGGTGAGAGTGAAGTCTGTCCCAGACCCACTGCCGCTGAACCTGT480    CTGAGATGCCAGAGGCCCTATTGGAAACACCATGTATGACCAGCCTTGGAGCCTGGCCAG540    GTTTGTGCTGGTACCAGGCTACGCGGCGGCTGCGAATGCTGTGACTGGACCTACAGGAGA600    AGGTGGCTCTTTCCCCTGGAGACAGAGACAGGGTGCCTGGAGACTGCGTGAGAACGATCT660    CACATCTGGCATCTGTAAGCCACAGCAGCAGCAACCCCAGGACCTGAGTGGGCACACCCA720    TGGTAAGCT729    (2) INFORMATION FOR SEQ ID NO:169:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3282 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169:    GGATCTAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTG60    AAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAG120    CTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGA180    GGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGA240    TCCGCCCTGCAGGGAAGGGCGGCACTGGGAAGTGGGCCAGGGCCAGGGACGTGACGTGGT300    GTGTGATCCCCTGTGTGTGTGTGGCGGCTGCAGGGGCACCCTTGTGAGAGGAGGGCTGGG360    TTTGTCTGAGCAGGTCAGCATGTGGAGAAGCTGCCGAGAGGCTCGTGGGCCTTGAGGTGC420    CGCGTGGGGCTCGTGGGGGCCTGTGTCCGAGGAGTGTTCACGTGTGCGAGGACCTTGCTC480    TGGTCTGGGTGCTGTGCAGTTCGCCCGGGTGAGGCTCCGTGTGTGAGGCGTGCACGTGTG540    TGTGTGGTGGCCGTGTGGCCGGCCAACCTCAGTGCGGGGGTTTTGTTGAACGGGTCTGGA600    CTGAGTGTGTGTGTGGGCATCTGCACCAGTCCCTCCACAGGGCCCGAGAGTGCATGTCCC660    CGGAGTCGGTTGTGTCCCCATGCGGGTGCGAGGCTGGGCAGGACAGCCAGGGGTTAGTGC720    CGTGGGGGTAGATGGGTGAGGGAGGGCCTGTCCCTACGCACATGGACTAGGCATGCCCCC780    GAGTGGGCATGGAGGTCGGAGGACAGGGCGCTCACAGAACAGGACAGTCTCCTACAGAGG840    CAGGGGCTGTGTGTCTGTCCCCAGGGGCTCCTAGGGCTTCCCGTGGCCCAGCCCAGGGCA900    GGTGCTGCTGGAGGGAGGGCAACGCTGGCAAATCCCCCACCCTGCCGAGGGCAGCCCCTG960    GCTGAGCCCCACCCTAGGCGGCCCAGGCACACCTGCACAGCCTGGGCCAGTGTGGGGACA1020    GTGGGACCCGCTCTGCCTCCCTCATGCCACTCAGGCCTCAGACTCGGCCTGACCCGTGGA1080    AAGAACCATCACAGTCTCGCAGGGGCCCAGGGCAGCGCTGGGTGCTTTATTTCCATGCTG1140    GGCGCCCGGGAAGTATGTACAGGGGGTACGTGCCAAGCATCCTCGTGCGACCGCGAGAGC1200    CCGGGGAGCGGGGGCTTGCCGGCCGTCGCACTCATTTACCCGGAGACAGGGAGAGGCTCT1260    TCTGCGTGTAGTGGTTGTGCAGAGCCTCATGCATCACGGAGCATGAGAAGACGTTCCCCT1320    GCTGCCACCTGCTCTTGTCCACGGTGAGCTTGCTGTAGAGGAAGAAGGAGCCGTCGGAGT1380    CCAGCACGGGAGGCGTGGTCTTGTAGTTGTTCTCCGGCTGCCCATTGCTCTCCCACTCCA1440    CGGCGATGTCGCTGGGATAGAAGCCTTTGACCAGGCAGGTCAGGCTGACCTGGTTCTTGG1500    TCAGCTCATCCCGGGATGGGGGCAGGGTGTACACCTGTGGTTCTCGGGGCTGCCCTGTAG1560    GGACAGAGGTTGGTACAGCGGTCACTCTCAGGGCAGAGGGTGGGCCGAGCCGGCCTCTGT1620    CCATGTGGCCCTCGCACCCCACGGGTCCCACCTTTGGCTTTGGAGATGGTTTTCTCGATG1680    GGGGCTGGGAGGGCTTTGTTGGAGACCTTGCACTTGTACTCCTTGCCATTCAGCCAGTCC1740    TGGTGCAGGACGGTGAGGACGCTGACCACACGGTACGTGCTGTTGTACTGCTCCTCCCGC1800    GGCTTTGTCTTGGCATTATGCACCTCCACGCCGTCCACGTACCAGTTGAACTTGACCTCA1860    GGGTCTTCGTGGCTCACGTCCACCACCACGCATGTGACCTCAGGGGTCCGGGAGATCATG1920    AGGGTGTCCTTGGGTTTTGGGGGGAAGAGGAAGACTGATGGTCCTCCCGCGGCCTCAGGT1980    GCTGAGGGAGAGATGGAGGTGGACGTGTCAGCACCCGGCTGGGGCCTGTCCCTGGATGCA2040    GGCTACTCTAGGGCACCTGTCCCGCCTTGAGCTGGAGGGCGAGGCCTGGGCTGGCTTACC2100    TGGGCACGGTGGGCATGTGTGAGTTTTGTCACAAGATTTGGGCTCTGCAGAGAGAAGATT2160    GGGAGTTACTCGAATCTGGGAGGAGAGAAGGTGTCCGAGCTGAGGGAGTGGAGAGTTTGG2220    CCTTTGGGGTGGGCTTAGGTCAGGGGCAGGGTCCTCCCGGATATGGCTCTTGGCAGGTCT2280    GAGCCCAGCACCTGCCCCTTTGTGTGCAGGGCCTGGGTTAGGGGCACCTAGCCTGTGCCT2340    GCCCAGAGCCTGGGGAAAAAGCCAGAAGACCCTCTCCCTGAGCATGAGTGGGGCGGGCAG2400    AGGCCTCCGGGTGAAGAGGCAGACGGGGCCTGCCTTGCTGCCCTGGACTGGGGCTGCATA2460    GCCGGGATGCGTCCAGGCAGGAGCGCTGAGCCTGGCTTCCAGCAGACACCCTCCCTCCCT2520    GTGCTGGCCTCTCACCAACTTTCTTGTCCACCTTGGTGTTGCTGGGCTTGTGATTCACGT2580    TGCAGATGTAGGTCTGGGTGCCCAAGCTGCTGGAGGGCACGGTCACCACGCTGCTGAGGG2640    AGTAGAGTCCTGAGGACTGTAGGACAGCCGGGAAGGTGTGCACGCCGCTGGTCAGGGCGC2700    CTGAGTTCCACGACACCGTCACCGGTTCGGGGAAGTAGTCCTTGACCAGGCAGCCCAGGG2760    CCGCTGTGCCCCCAGAGGTGCTCTTGGAGGAGGGTGCCAGGGGGAAGACCGATGGGCCCT2820    TGGTGGAAGCTGAGCTCACGATGACCGTGGTTCCTTTGCCCCAGACGTCCATATAATAAT2880    TGTCCTGGGGAGAATCATCCCAACTATATGGCCCCACTCTCGCACAATAATAAACAGCCG2940    TGTCTGCAGACCTGAGGCTCCTCAACTCCATGTAGGCTGTGTTCGCGGATGTGTCCGCGG3000    TAAAGGTGACTCTGTCCTGGAACTTCGCTGAAAATTCTTTGTTTCCGTTGTAAGGATTGA3060    TCCATCCCATCCACTCAAACCTCTGTCCGGGGGCCTGGCGCACCCAATGAATAACAAAGT3120    TACTGAATCTGTATCCAGAAGCCTGACAAGAAACCTTCACTGAGGCCCCAGGCTTCTTCA3180    CCTCAGCCCCGGACTGAACCAGCTGAACCTGGGAGTGGACACCTGTAGTTACTGACAGGA3240    AGAAGAGAAAGACCCAGCTCCATTCCATGGTGGCGGCGAATT3282    (2) INFORMATION FOR SEQ ID NO:170:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13254 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: circular    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:170:    TTCTCTAGAATTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCGATCTCAGGCCCT60    GATGGGTGACTTCGCAGGCGTAGACTTTGTGTTTCTCGTAGTCTGCTTTGCTCAGCGTCA120    GGGTGCTGCTGAGGCTGTAGGTGCTGTCCTTGCTGTCCTGCTCTGTGACACTCTCCTGGG180    AGTTACCCGATTGGAGGGCGTTATCCACCTTCCACTGTACTTTGGCCTCTCTGGGATAGA240    AGTTATTCAGCAGGCACACAACAGAGGCAGTCCCAGATTTCAACTGCTCATCAGATGGCG300    GGAAGATGAAGACAGATGGTGCAGGCACAGTTCGTTTCCTCTCCAGTTTGGTCCCCTGGC360    CAAAAGTGTACGAGGAGGCACCATAGACCTGACAGTAGTACAGTGCAAAGTCTTCAGGCT420    CCACTCTGGTGATGGTGAGAGTGAAGTCTGTCCCAGACCCACTGCCGCTGAACCTGTCTG480    AGATGCCAGAGGCCCTATTGGAAACACCATGTATGACCAGCCTTGGAGCCTGGCCAGGTT540    TGTGCTGGTACCAGGCTACGCGGCGGCTGCGAATGCTGTGACTGGACCTACAGGAGAAGG600    TGGCTCTTTCCCCTGGAGACAGAGACAGGGTGCCTGGAGACTGCGTGAGAACGATCTCAC660    ATCTGGCATCTGTAAGCCACAGCAGCAGCAACCCCAGGACCTGAGTGGGCACACCCATGG720    TAAGCTTCGTGTCAAGGACGGTGACTGCAGAAAAGACCCATGGAAAGGAACAGTCTGTTA780    GTCTGTCAGCTATTATGTCTGGTGGCGCGCGCGGCAGCAACGAGTACTGCTCAGACTACA840    CTGCCCTCCACCGTTAACAGCACCGCAACGGGAGTTACCTCTGACTCTTATCAGAACACA900    ACAACTCAGCTGCCTGCATCTTCTTCTGCCGCTGCCTTAAGTCTTCCAAATGCGTCAGCG960    GTGCAAGCCCGCTCCCCGAGCTCATTTTCAGACACATACCCTACCGCCACGGCCTTGTGC1020    GGCACACTGGTGGTGGTGGGCATCGTGCTGTGCCTAAGTCTGGCCTCCACTGTTAGGAGC1080    AAGGAGCTGCCGAGCGACCATGAGTCGCTGGAGGCATGGGAGCAGGGCTCGGATGTAGAA1140    GCTCCGCCGCTACCGGAGAAGAGCCCATGTCCGGAACACGTACCCGAGATTCGCGTGGAG1200    ATCCCACGTTATGTTTAATAAAAACTGCGGGCACTGGGGACGGTGGTGTTGTATATGTGA1260    ATTTGTAAATAATAAATGAGACCCCATCCTGTAAAAATACAGAGTCCGTGTCAGTCTCTG1320    AAGGACAGTGTATTGGCATATAGCCAATAAAGAGAGTTGTGGCAAAGAGCCATGTTATGG1380    ATTAGTAATGGAAAGTATCGTCACCAATAGGGGAGTGGTCAATAATGGTCAATAACCCAC1440    ACCTATAGGCTAAGCTATACCATCACCTATAACATGAGGAAGCGGGGGTGTATAGACCCC1500    AAGCCAAAAACAGTATAGCATGCATAAGAAGCCAAGGGGGTGGGCCTATAGACTCTATAG1560    GCGGTACTTACGTCACTCTTGGCACGGGGAATCCGCGTTCCAATGCACCGTTCCCGGCCG1620    CGGAGGCTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCGTGGATGGCGTCTC1680    CAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGC1740    CTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGA1800    TTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGT1860    GAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATA1920    GCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGG1980    GCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATA2040    TGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGT2100    CAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGG2160    CGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAAC2220    TCCATATATGGGCTATGAACTAATGACCCCGTAATTGATTACTATTAATAACTAGTCAAT2280    AATCAATGTCAACATGGCGGTAATGTTGGACATGAGCCAATATAAATGTACATATTATGA2340    TATGGATACAACGTATGCAATGGCCAATAGCCAATATTGATTTATGCTATATAACCAATG2400    AATAATATGGCTAATGGCCAATATTGATTCAATGTATAGATCGATATGCATTGGCCATGT2460    GCCAGCTTGATGTCGCCTCTATCGGCGATATAGCCTCATATCGTCTGTCACCTATATCGA2520    AACTGCGATATTTGCGACACACAGAATCGCCCAAGTCACCAAAGTCGTCTATCGCCATCC2580    CCCGTAAACGATATAAGCGCTATCGCCAGATATCGCGTATGCCCAAAAATCACTTTTGGA2640    AAAATGGCGATATCAGTTACACAGAAACTCACATCGGCGACATTTTCAATATGCCATATT2700    TTCAAATATCGATTTTTCCAATATCGCCATCTCTATCGGCGATAAACACCACTATCGCGC2760    GACATGAATTTAGTCGGCGACAGAAATCTCAAAACGCGTATTTCGGACAAACACACATTT2820    TATTATTCACTGCAGTCACCGTCCTTGACACGAAGCTAGAGATCCAGACATGATAAGATA2880    CATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGA2940    AATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAA3000    CAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAG3060    CAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCTCTAGTCAAGGCACTATAC3120    ATCAAATATTCCTTATTAACCCCTTTACAAATTAAAAAGCTAAAGGTACACAATTTTTGA3180    GCATAGTTATTAATAGCAGACACTCTATGCCTGTGTGGAGTAAGAAAAAACAGTATGTTA3240    TGATTATAACTGTTATGCCTACTTATAAAGGTTACAGAATATTTTTCCATAATTTTCTTG3300    TATAGCAGTGCAGCTTTTTCCTTTGTGGTGTAAATAGCAAAGCAAGCAAGAGTTCTATTA3360    CTAAACACAGCATGACTCAAAAAACTTAGCAATTCTGAAGGAAAGTCCTTGGGGTCTTCT3420    ACCTTTCTCTTCTTTTTTGGAGGAGTAGAATGTTGAGAGTCAGCAGTAGCCTCATCATCA3480    CTAGATGGCATTTCTTCTGAGCAAAACAGGTTTTCCTCATTAAAGGCATTCCACCACTGC3540    TCCCATTCATCAGTTCCATAGGTTGGAATCTAAAATACACAAACAATTAGAATCAGTAGT3600    TTAACACATTATACACTTAAAAATTTTATATTTACCTTAGAGCTTTAAATCTCTGTAGGT3660    AGTTTGTCCAATTATGTCACACCACAGAAGTAAGGTTCCTTCACAAAGATCTCTCTGGGG3720    CGGGGTGGGATGAACTAGGAAAGGCTCAAGATCACTCAAAGTCTAATTAGTTTTTGTATT3780    GGAAGGGCTCGTGGCCAGTCTCATTGAGAAGGCATGTGCGGACGATGGCTTCTGTCACTG3840    CAAAGGGGTCACAATTGGCAGAGGGGCCGCGGTCTTCAAAGTAACCTTTCTTCTCCTGGC3900    CGACAGTCCGGGGAATGCGGATGCTGGCACTGCGATTGGCGACACCAGCAGAAAAGTCGT3960    TGATGTTGGACGTTTCGTGGAACCCAGTCAGACCACGGGCATTGTCCAGGCCCCCCTTGG4020    GATCGTAGGCTCGAATGTGGTACCGGTGCCGCTTGCTTAGTTTCTCGATGGCCTCCTCGA4080    TGTGCTTCAGACCATTCTCCTCCCGCATGGCCTTGGTGCTAAAGTTGGTATGGCAGCCTG4140    CACCATTCCAGTTCCCAGGAATGGGCTTGGGGTCAAAGGTTGCTATTACCCCAAAGTCTT4200    CACATACTCGATGAAGATGAAACGGGCCACCCAGAGATGATCTCCCATGCGGATTCCTTC4260    ACAGGGTCCTATTTGGAGTTCCCACTGGGCAGGCATGACCTCAGCATTTGTTCCTGTAAT4320    CTTGACCCCAGCATACAAGCAGGCGCGGTAGTGAGCCTCCACGATATCCCTGCCATAGGC4380    TTTGTCTGCGCCCACACCACAGTAATACGGACCTTGGGGCCCAGGAAAGCCATTGGAAGG4440    CCAACCAAAAGGGTGCCCATCTGTTCCCATCAGAGTATACTCCTGTTCCATTCCAAACCA4500    GGGGTGCTGGTTGCTCACCATGTCCATTATCCGTTTACACGAGTGCCTTAAATTGGTCTC4560    TGCAGGCTTCCGGTTGTACTTGAAAACTTCACAGAACACCAGCTTGTTGGGATCTCTGCG4620    GAAGGGGTCCCGAAACATGGCAACAGGGCTGAGATACATGTCACTGTTGGAGCCCTCAGA4680    CTGAAAGGTACTAGAGCCATCAAAATTCCACTCAGGTAACTCTTCTACACACTTGGGCTC4740    ACAGTCCAGGGTGCGGGTTTTGCAGCGCAGTCCTTCTCCAGTACCATCAACCCAGATATA4800    CATGGCTTGGACTTTCTCACCCTGGGGCAGGCACAAGTACATTTGCTTGATGTTTTTGTT4860    CAAGTGGGAACTTGCTGAGGTGGCCATGGTGGAAGGTGCTCTGAGCGGTGGCCCCAAGCT4920    AGTTTTTGCAAAAGCCTAGGCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAG4980    AGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGGGGCGGAG5040    AATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATGG5100    TTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGTTGCT5160    GACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCA5220    CACCCTAACTGACACACATTCCACAGCCGGATCTAGCTTGGCTGCCAAAGCCGCAAGGAA5280    TTTACCAACCTTCTTAAACATAAAGTGTCTCCTTATAAACGCAGAAAGGCCCACCCGAAG5340    GTGAGCCAGTGTGATTACATTTTCTCTTGAGGGTTGTCCTCGGTGCCACGGAACATTACG5400    AACGATGGGTGCCGCAAAGAGCCATCAGGGCCTCGTGATACGCCTATTTTTATAGGTTAA5460    TGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGG5520    AACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATA5580    ACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCG5640    TGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAAC5700    GCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACT5760    GGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGAT5820    GAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGA5880    GCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCAC5940    AGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCAT6000    GAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAAC6060    CGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCT6120    GAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAAC6180    GTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGA6240    CTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTG6300    GTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACT6360    GGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAAC6420    TATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTA6480    ACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT6540    TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGA6600    GTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCC6660    TTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGT6720    TTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGC6780    GCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTC6840    TGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGG6900    CGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCG6960    GTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA7020    ACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGC7080    GGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGG7140    GGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCG7200    ATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCCG7260    AGGTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAG7320    GCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCC7380    CCCGGCCACGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGG7440    CGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTG7500    GCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCTAGACATGATAAGATACA7560    TTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAA7620    TTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACA7680    ACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCA7740    AGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCCGCCCTGCAGGGAAGGGCGGC7800    ACTGGGAAGTGGGCCAGGGCCAGGGACGTGACGTGGTGTGTGATCCCCTGTGTGTGTGTG7860    GCGGCTGCAGGGGCACCCTTGTGAGAGGAGGGCTGGGTTTGTCTGAGCAGGTCAGCATGT7920    GGAGAAGCTGCCGAGAGGCTCGTGGGCCTTGAGGTGCCGCGTGGGGCTCGTGGGGGCCTG7980    TGTCCGAGGAGTGTTCACGTGTGCGAGGACCTTGCTCTGGTCTGGGTGCTGTGCAGTTCG8040    CCCGGGTGAGGCTCCGTGTGTGAGGCGTGCACGTGTGTGTGTGGTGGCCGTGTGGCCGGC8100    CAACCTCAGTGCGGGGGTTTTGTTGAACGGGTCTGGACTGAGTGTGTGTGTGGGCATCTG8160    CACCAGTCCCTCCACAGGGCCCGAGAGTGCATGTCCCCGGAGTCGGTTGTGTCCCCATGC8220    GGGTGCGAGGCTGGGCAGGACAGCCAGGGGTTAGTGCCGTGGGGGTAGATGGGTGAGGGA8280    GGGCCTGTCCCTACGCACATGGACTAGGCATGCCCCCGAGTGGGCATGGAGGTCGGAGGA8340    CAGGGCGCTCACAGAACAGGACAGTCTCCTACAGAGGCAGGGGCTGTGTGTCTGTCCCCA8400    GGGGCTCCTAGGGCTTCCCGTGGCCCAGCCCAGGGCAGGTGCTGCTGGAGGGAGGGCAAC8460    GCTGGCAAATCCCCCACCCTGCCGAGGGCAGCCCCTGGCTGAGCCCCACCCTAGGCGGCC8520    CAGGCACACCTGCACAGCCTGGGCCAGTGTGGGGACAGTGGGACCCGCTCTGCCTCCCTC8580    ATGCCACTCAGGCCTCAGACTCGGCCTGACCCGTGGAAAGAACCATCACAGTCTCGCAGG8640    GGCCCAGGGCAGCGCTGGGTGCTTTATTTCCATGCTGGGCGCCCGGGAAGTATGTACAGG8700    GGGTACGTGCCAAGCATCCTCGTGCGACCGCGAGAGCCCGGGGAGCGGGGGCTTGCCGGC8760    CGTCGCACTCATTTACCCGGAGACAGGGAGAGGCTCTTCTGCGTGTAGTGGTTGTGCAGA8820    GCCTCATGCATCACGGAGCATGAGAAGACGTTCCCCTGCTGCCACCTGCTCTTGTCCACG8880    GTGAGCTTGCTGTAGAGGAAGAAGGAGCCGTCGGAGTCCAGCACGGGAGGCGTGGTCTTG8940    TAGTTGTTCTCCGGCTGCCCATTGCTCTCCCACTCCACGGCGATGTCGCTGGGATAGAAG9000    CCTTTGACCAGGCAGGTCAGGCTGACCTGGTTCTTGGTCAGCTCATCCCGGGATGGGGGC9060    AGGGTGTACACCTGTGGTTCTCGGGGCTGCCCTGTAGGGACAGAGGTTGGTACAGCGGTC9120    ACTCTCAGGGCAGAGGGTGGGCCGAGCCGGCCTCTGTCCATGTGGCCCTCGCACCCCACG9180    GGTCCCACCTTTGGCTTTGGAGATGGTTTTCTCGATGGGGGCTGGGAGGGCTTTGTTGGA9240    GACCTTGCACTTGTACTCCTTGCCATTCAGCCAGTCCTGGTGCAGGACGGTGAGGACGCT9300    GACCACACGGTACGTGCTGTTGTACTGCTCCTCCCGCGGCTTTGTCTTGGCATTATGCAC9360    CTCCACGCCGTCCACGTACCAGTTGAACTTGACCTCAGGGTCTTCGTGGCTCACGTCCAC9420    CACCACGCATGTGACCTCAGGGGTCCGGGAGATCATGAGGGTGTCCTTGGGTTTTGGGGG9480    GAAGAGGAAGACTGATGGTCCTCCCGCGGCCTCAGGTGCTGAGGGAGAGATGGAGGTGGA9540    CGTGTCAGCACCCGGCTGGGGCCTGTCCCTGGATGCAGGCTACTCTAGGGCACCTGTCCC9600    GCCTTGAGCTGGAGGGCGAGGCCTGGGCTGGCTTACCTGGGCACGGTGGGCATGTGTGAG9660    TTTTGTCACAAGATTTGGGCTCTGCAGAGAGAAGATTGGGAGTTACTCGAATCTGGGAGG9720    AGAGAAGGTGTCCGAGCTGAGGGAGTGGAGAGTTTGGCCTTTGGGGTGGGCTTAGGTCAG9780    GGGCAGGGTCCTCCCGGATATGGCTCTTGGCAGGTCTGAGCCCAGCACCTGCCCCTTTGT9840    GTGCAGGGCCTGGGTTAGGGGCACCTAGCCTGTGCCTGCCCAGAGCCTGGGGAAAAAGCC9900    AGAAGACCCTCTCCCTGAGCATGAGTGGGGCGGGCAGAGGCCTCCGGGTGAAGAGGCAGA9960    CGGGGCCTGCCTTGCTGCCCTGGACTGGGGCTGCATAGCCGGGATGCGTCCAGGCAGGAG10020    CGCTGAGCCTGGCTTCCAGCAGACACCCTCCCTCCCTGTGCTGGCCTCTCACCAACTTTC10080    TTGTCCACCTTGGTGTTGCTGGGCTTGTGATTCACGTTGCAGATGTAGGTCTGGGTGCCC10140    AAGCTGCTGGAGGGCACGGTCACCACGCTGCTGAGGGAGTAGAGTCCTGAGGACTGTAGG10200    ACAGCCGGGAAGGTGTGCACGCCGCTGGTCAGGGCGCCTGAGTTCCACGACACCGTCACC10260    GGTTCGGGGAAGTAGTCCTTGACCAGGCAGCCCAGGGCCGCTGTGCCCCCAGAGGTGCTC10320    TTGGAGGAGGGTGCCAGGGGGAAGACCGATGGGCCCTTGGTGGAAGCTGAGCTCACGATG10380    ACCGTGGTTCCTTTGCCCCAGACGTCCATATAATAATTGTCCTGGGGAGAATCATCCCAA10440    CTATATGGCCCCACTCTCGCACAATAATAAACAGCCGTGTCTGCAGACCTGAGGCTCCTC10500    AACTCCATGTAGGCTGTGTTCGCGGATGTGTCCGCGGTAAAGGTGACTCTGTCCTGGAAC10560    TTCGCTGAAAATTCTTTGTTTCCGTTGTAAGGATTGATCCATCCCATCCACTCAAACCTC10620    TGTCCGGGGGCCTGGCGCACCCAATGAATAACAAAGTTACTGAATCTGTATCCAGAAGCC10680    TGACAAGAAACCTTCACTGAGGCCCCAGGCTTCTTCACCTCAGCCCCGGACTGAACCAGC10740    TGAACCTGGGAGTGGACACCTGTAGTTACTGACAGGAAGAAGAGAAAGACCCAGCTCCAT10800    TCCATGGTGGCGGCGAATTCGAGCTCGCCCGGGGATCGATCCTCTAGAGTCGATCGACCT10860    GCAGCCCAAGCTTCGTGTCAAGGACGGTGACTGCAGAAAAGACCCATGGAAAGGAACAGT10920    CTGTTAGTCTGTCAGCTATTATGTCTGGTGGCGCGCGCGGCAGCAACGAGTACTGCTCAG10980    ACTACACTGCCCTCCACCGTTAACAGCACCGCAACGGGAGTTACCTCTGACTCTTATCAG11040    AACACAACAACTCAGCTGCCTGCATCTTCTTCTGCCGCTGCCTTAAGTCTTCCAAATGCG11100    TCAGCGGTGCAAGCCCGCTCCCCGAGCTCATTTTCAGACACATACCCTACCGCCACGGCC11160    TTGTGCGGCACACTGGTGGTGGTGGGCATCGTGCTGTGCCTAAGTCTGGCCTCCACTGTT11220    AGGAGCAAGGAGCTGCCGAGCGACCATGAGTCGCTGGAGGCATGGGAGCAGGGCTCGGAT11280    GTAGAAGCTCCGCCGCTACCGGAGAAGAGCCCATGTCCGGAACACGTACCCGAGATTCGC11340    GTGGAGATCCCACGTTATGTTTAATAAAAACTGCGGGCACTGGGGACGGTGGTGTTGTAT11400    ATGTGAATTTGTAAATAATAAATGAGACCCCATCCTGTAAAAATACAGAGTCCGTGTCAG11460    TCTCTGAAGGACAGTGTATTGGCATATAGCCAATAAAGAGAGTTGTGGCAAAGAGCCATG11520    TTATGGATTAGTAATGGAAAGTATCGTCACCAATAGGGGAGTGGTCAATAATGGTCAATA11580    ACCCACACCTATAGGCTAAGCTATACCATCACCTATAACATGAGGAAGCGGGGGTGTATA11640    GACCCCAAGCCAAAAACAGTATAGCATGCATAAGAAGCCAAGGGGGTGGGCCTATAGACT11700    CTATAGGCGGTACTTACGTCACTCTTGGCACGGGGAATCCGCGTTCCAATGCACCGTTCC11760    CGGCCGCGGAGGCTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCGTGGATGG11820    CGTCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGT11880    ACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCC11940    CGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAAT12000    CCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATG12060    GTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATG12120    TACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTT12180    GGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCAT12240    TGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTC12300    AATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACG12360    CGGAACTCCATATATGGGCTATGAACTAATGACCCCGTAATTGATTACTATTAATAACTA12420    GTCAATAATCAATGTCAACATGGCGGTAATGTTGGACATGAGCCAATATAAATGTACATA12480    TTATGATATGGATACAACGTATGCAATGGCCAATAGCCAATATTGATTTATGCTATATAA12540    CCAATGAATAATATGGCTAATGGCCAATATTGATTCAATGTATAGATCGATATGCATTGG12600    CCATGTGCCAGCTTGATGTCGCCTCTATCGGCGATATAGCCTCATATCGTCTGTCACCTA12660    TATCGAAACTGCGATATTTGCGACACACAGAATCGCCCAAGTCACCAAAGTCGTCTATCG12720    CCATCCCCCGTAAACGATATAAGCGCTATCGCCAGATATCGCGTATGCCCAAAAATCACT12780    TTTGGAAAAATGGCGATATCAGTTACACAGAAACTCACATCGGCGACATTTTCAATATGC12840    CATATTTTCAAATATCGATTTTTCCAATATCGCCATCTCTATCGGCGATAAACACCACTA12900    TCGCGCGACATGAATTTAGTCGGCGACAGAAATCTCAAAACGCGTATTTCGGACAAACAC12960    ACATTTTATTATTCACTGCAGTCACCGTCCTTGACACGAAGCTAGAGATCCAGACATGAT13020    AAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTAT13080    TTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGT13140    TAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTT13200    TTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTAATGATCAATGAA13254    __________________________________________________________________________

What is claimed is:
 1. A human monoclonal antibody that immunoreactswith human immunodeficiency virus (HIV) glycoprotein gp120, neutralizesHIV, and is produced by the cell line designated MT12 having A.T.C.C.Accession Number
 69079. 2. A human monoclonal antibody comprising thevariable region amino acid sequence of the human monoclonal antibodyproduced by the cell line designated MT12 having A.T.C.C. AccessionNumber
 69079. 3. A cell line designated MT12 having A.T.C.C. AccessionNumber 69079.